Ferroportin1 nucleic acids and proteins

ABSTRACT

Positional cloning has been carried out to identify the gene responsible for the hypochromic anemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a novel multiple-transmembrane domain protein, expressed in the yolk sac. Zebrafish ferroportin1 is required for the transport of iron from maternally-derived yolk stores to the circulation, and functions as an iron exporter when expressed in  Xenopus  oocytes. Human and mouse homologs of the ferroportin1 gene have been identified. The invention includes isolated polynucleotides, vectors and host cells comprising nucleotide sequences encoding Ferroportin1 proteins and variants thereof, including those having iron transport function. The invention also includes polypeptides encoded by ferroportin1 genes and variants of such polypeptides, and fusion polypeptides comprising a Ferroportin1 or a portion thereof. Methods to produce a Ferroportin1, methods to produce antibodies to a Ferroportin1 and methods to identify agents binding to a Ferroportin1, which can be inhibitors or enhancers of Ferroportin1 iron transport activity, are also described. Inhibitors of Ferroportin1 activity can be used in a therapy for hemochromatosis.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/715,927, filed Nov. 17, 2000, which is a continuation-in-part of U.S.application Ser. No. 09/567,672, filed May 9, 2000 which claims thebenefit of U.S. Provisional Application No. 60/133,382, filed on May 10,1999. The entire teachings of the above applications are incorporatedherein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant R01DL53298-02 from the National Institutes for Health. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Defects in iron absorption and utilization lead to iron deficiency andoverload disorders. Adult mammals absorb iron through the duodenum,whereas embryos obtain iron through placental transport. Iron uptakefrom the intestinal lumen through the apical surface of polarizedduodenal enterocytes is mediated by the divalent metal transporter, DMT1(Fleming, M. D., et al., Nature Genet., 16:383-386, 1997; Gunshin, H.,et al., Nature, 388:482-488, 1997; Andrews, N. C., N. Engl. J. Med.,341:1986-1995, 1999). A second transporter has been postulated to exportiron across the basolateral surface to the circulation. The function ofthis iron transporting protein may be perturbed in mammalian disordersof iron deficiency or overload. Drugs to alter the function of this irontransporting protein may be useful to treat such diseases ashemochromatosis and some forms of anemia.

SUMMARY OF THE INVENTION

The invention relates to a number of nucleic acids, wherein the nucleicacids have SEQ ID NO:1, 3, 5 or 7 as described herein or the nucleicacids have nucleotide sequences related to those given specifically bySEQ ID NO by properties of hybridization, or by varying extents ofidentity, or by varying degrees of similarity as can be determined by acomputer program designed for the purpose of comparing nucleotide oramino acid sequences. SEQ ID NO:1 is the nucleotide sequence of a cDNAencoding a zebrafish ferroportin1; SEQ ID NO:3 is the nucleotidesequence of a cDNA encoding a mouse ferroportin1; SEQ ID NO:5 is thenucleotide sequence of a cDNA encoding a human ferroportin1. SEQ ID NO:7is the nucleotide sequence of a genomic DNA comprising the introns andexons of a human ferroportin1 gene. Also part of the invention arecontiguous portions of any of the above nucleic acids, nucleic acidsencoding any of the amino acid sequences described herein and nucleicacids encoding polypeptides which are variants of the Ferroportin1proteins described herein by amino acid sequence. Further nucleic acidswhich are part of the invention are those encoding a fusion polypeptidecomprising a Ferroportin1 or a portion of a Ferroportin1.

Related to the isolated nucleic acids are vectors and host cellscomprising nucleotide sequences identical to the isolated nucleic acidsof the invention. In some cases, regulatory sequences can be operablylinked to coding regions to allow expression of a gene. Such cells canbe maintained under conditions in which the gene is expressed and theencoded polypeptide is produced. The polypeptide can be purified by oneor more steps to increase the proportion of polypeptide in the milieu ofmedium and material of cellular origin, thereby producing isolatedpolypeptide.

The term regulatory element refers to a genetic element which controlssome aspect of the expression of nucleic acid sequences. For example, apromoter is a regulatory element which facilitates the initiation oftranscription of an operably linked coding region. Other regulatoryelements are splicing signals, polyadenylation signals, terminationsignals and enhancers, for instance.

The invention also includes Ferroportin1 proteins, for example, thosehaving amino acid sequence SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6,proteins which are naturally occurring mutants or variants of thoseproteins characterized by those specific amino acid sequences, andmutants and variants of those proteins identified as having the specificamino acid sequence SEQ ID NO:2, 4, or 6 that are produced by laboratorymanipulations of the nucleic acids encoding a Ferroportin1. Also withinthe invention are contiguous portions of any of the polypeptides withSEQ ID NO:2, 4, or 6, or portions of such mutants or variants of thepolypeptides described herein as containing amino acid substitutions, ordescribed herein as having a certain percent identity or similarity toanother sequence in a comparison. A further embodiment of the inventionis a fusion polypeptide, which can comprise a Ferroportin1 offull-length amino acid sequence, as in SEQ ID NO:2, SEQ ID NO:4 or SEQID NO:6, or a contiguous portion thereof, or can comprise any of themutants or variant polypeptides, or portions thereof, as describedherein, for example by their amino acid sequence identity or similarityto an amino acid sequence identified by SEQ ID NO, or by their activity(e.g., iron transport function) or property of binding to antibodiesproduced by immunizing an animal with a Ferroportin1.

Antibodies that bind to one or more Ferroportin1 proteins are also anaspect of the invention. Antibodies to a Ferroportin1 of one or morespecies can be produced, for example, by introducing into an animalwhich is not the source of the Ferroportin1 immunogen a Ferroportin1 oran immunogenic portion thereof in a suitable medium, which can includesuch substances as stabilizing agents and adjuvant. Other known methodscan be used to make hybridomas producing monoclonal antibodies that bindto one or more Ferroportin1 proteins, as isolated, or as they exist in acell membrane.

Other aspects of the invention include methods for identifying agentswhich bind to a Ferroportin1 (or to a mutant, variant, Ferroportin1fusion protein or a contiguous portion of any of the foregoing) by stepsthat include contacting the agent with the isolated protein underconditions appropriate for binding of the agent to the isolated protein,and detecting a resulting agent-protein complex. Similar methods can beused to identify an agent which is an inhibitor or an enhancer of afunction of a Ferroportin1 protein, where the steps can be thefollowing: (a) combining (1) said isolated protein; (2) the ligand ofsaid protein; and (3) a candidate agent to be assessed for its abilityto inhibit interaction between said protein of (1) and the ligand of(2), under conditions appropriate for interaction between the saidprotein of (1) and the ligand of (2); (b) determining the extent towhich said protein of (1) and the ligand of (2) interact; and (c)comparing the extent determined in (b) with the extent to whichinteraction of said protein of (1) and the ligand of (2) occurs in theabsence of the candidate agent to be assessed and under the sameconditions appropriate for interaction of said protein of (1) with theligand of (2); wherein if the extent to which interaction of saidprotein of (1) and the ligand of (2) occurs is less in the presence ofthe candidate agent than in the absence of the candidate agent, thecandidate agent is an agent which inhibits interaction between saidprotein and the ligand of said protein. If greater export of the ironfrom the test cells compared to export of the iron from the controlcells is observed, this is indicative that the agent is an enhancer ofiron export by said protein. An agent can be further tested for itseffect on a Ferroportin1 protein in an animal, if the following stepsare carried out: a) administering the agent to one or more test animals;b) measuring exogenously supplied iron in one or more samples of tissueor bodily fluid from said test animals; c) measuring exogenouslysupplied iron in one or more comparable samples of tissue or bodilyfluid from suitable control animals; and d) comparing the iron of b)with the iron of c); whereby, lower iron in step b) than in step c) isindicative that the agent is an inhibitor of said protein. An inhibitorof the iron transport function of a human Ferroportin1 can be used in amethod for treating hemochromatosis in a human, said method comprisingadministering to the human an inhibitor of Ferroportin1 iron transportfunction, or such inhibitor can be used in a method for treating adisease or medical disorder resulting from oxidative damage in a mammal,said method comprising administering to the mammal an inhibitor ofFerroportin1 iron transport function. Enhancers of a Ferroportin1 can beused in a method for treating iron deficiency anemia in a mammal, saidmethod comprising administering to the mammal an enhancer ofFerroportin1 iron transport function.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingembodiments of the present invention.

FIG. 1 is a map of the weh locus showing positional cloning of theweissherbst gene. The weh locus is depicted by a thick black bar justdistal to the AFLP marker 136. Below the map is an enlarged view of theweh locus that depicts the BAC and PAC genomic clones identified by achomosomal walk in an analysis of 3873 meioses. Genotyping of a total of1783 meioses from haploid animals and 2090 meioses from diploid mutantsnarrowed the critical interval containing the gene to the PAC clones211O13 and 170G3. The numbers of recombination events identified on theproximal side (circles) and distal side (squares) of the weh locus areindicated.

FIG. 2 is an amino acid sequence alignment of zebrafish, human and mouseFerroportin1 (FPN1). The initiator methionine in all three species wasestablished by the presence of upstream, in-frame stop codons. Shadingindicates identical amino acids. Bars under sequence indicate predictedtransmembrane domains. The mutations identified in the weh^(tp85c) andweh^(th238) alleles are indicated by black circles below the affectedamino acids.

FIG. 3 is a bar graph showing the measurement of iron efflux fromXenopus oocytes. Oocytes expressing either DMT1 alone or DMT1 and FPN1were loaded with ⁵⁵Fe by incubation in uptake buffer containing 60 μM⁵⁵FeCl₂. Efflux from individual oocytes was measured by incubation ofoocytes in 500 μl of efflux buffer with or without 20 mg/mlapo-transferrin (−apoTfr and +apoTfr). After efflux, the total ⁵⁵Fecontent of both the efflux solution and the individual oocytes wasmeasured by scintillation counting. The data are expressed as an average(n=6) of the ratio of the pmols of efflux to the pmols of uptake peroocyte.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

Iron is required for many cellular processes, but it can also be toxicwhen present in excess. Thus, iron homeostasis must be strictlymaintained. Studies described herein employed zebrafish genetics toidentify the multiple-transmembrane domain protein Ferroportin1, an ironexport protein. In the mammalian yolk sac and placenta, Ferroportin1 mayplay an important conserved role in the transport of iron from thematernal to the embryonic circulation. In adults, Ferroportin1 is likelyto function in iron transport at the basolateral surface of duodenalenterocytes. In disorders such as iron deficiency or overload, tissuesrespond by altering normal iron utilization. Ferroportin1 could beinvolved in the pathophysiology of iron deficiency anemias or ironoverload syndromes, such as hemochromatosis.

As described herein, Ferroportin1 refers to an evolutionarily conservedfamily of proteins that mediate the transport of iron out of cells. Thefamily includes proteins which are conserved at least as widely as fromzebrafish to humans and exhibit very different expression patterns intissues. Specific embodiments described include Ferroportin1 proteinsfrom mice, humans and zebrafish which have been shown to be functionaliron transporters. The term Ferroportin1 can refer to other proteinssharing at least about 70% sequence similarity, more preferably at leastabout 80% sequence similarity, and still more preferably, at least about90% sequence similarity, and most preferably, at least about 95%sequence similarity.

One aspect of the invention relates to isolated nucleic acids orpolynucleotides that encode a Ferroportin1 as described herein, such asthose Ferroportin1 proteins having an amino acid sequence SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:6 and nucleic acids closely related thereto asdescribed herein.

Using the information provided herein, such as a nucleic acid sequenceset forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, a nucleic acid ofthe invention encoding a Ferroportin1 polypeptide may be obtained usingstandard cloning and screening methods, such as those for cloning andsequencing cDNA library fragments, followed by obtaining a full lengthclone. For example, to obtain a nucleic acid of the invention, a libraryof clones of cDNA of a species of animal can be probed with a labeledoligonucleotide, such as a radiolabeled oligonucleotide, preferablyabout 17 nucleotides or longer, derived from a partial sequence. Clonescarrying DNA identical to that of the probe can then be distinguishedusing stringent (also, “high stringency”) hybridization conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full length sequence.Suitable techniques are described, for example, in Current Protocols inMolecular Biology (F. M. Ausubel et al., eds.), containing supplementsthrough Supplement 49, 2000, John Wiley and Sons, Inc., especiallychapters 5, 6 and 7.

Embodiments of the invention include isolated nucleic acid moleculescomprising any of the following nucleotide sequences: 1.) a nucleotidesequence which encodes a protein comprising the amino acid sequence ofhuman Ferroportin1 (SEQ ID NO:6), the amino acid sequence of mouseFerroportin1 (SEQ ID NO:4), or the amino acid sequence of zebrafishFerroportin1 (SEQ ID NO:2); 2.) nucleotide sequences of humanferroportin1, mouse ferroportin1, or zebrafish ferroportin1; 3.) anucleotide sequence which is complementary to the nucleotide sequence ofhuman ferroportin1 (SEQ ID NO:5), mouse ferroportin1 (SEQ ID NO:3),zebrafish ferroportin1 (SEQ ID NO:1); 4.) a nucleotide sequence whichconsists of the coding region of human ferroportin1 (within SEQ IDNO:5), the coding region of mouse ferroportin1 (within SEQ ID NO:3), orthe coding region of zebrafish ferroportin1 (within SEQ ID NO:1).

The invention further relates to nucleic acids (nucleic acid moleculesor polynucleotides) having nucleotide sequences identical over theirentire length to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO: 5. It furtherrelates to DNA, which due to the degeneracy of the genetic code, encodesa Ferroportin1 protein whose amino acid sequence is provided herein.Also provided by the invention are nucleic acids having the codingsequences for the mature polypeptides or fragments in reading frame withother coding sequences, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro-protein sequence. The nucleic acidsof the invention encompass nucleic acids that include a singlecontinuous region or discontinuous regions encoding the polypeptide,together with additional regions, that may also contain coding ornon-coding sequences. The nucleic acids may also contain non-codingsequences, including, for example, but not limited to, non-coding 5′ and3′ sequences, such as the transcribed, non-translated sequences,termination signals, ribosome binding sites, sequences that stabilizemRNA, introns, polyadenylation signals, and additional coding sequenceswhich encode additional amino acids.

The nucleic acid molecules of the invention can comprise, in addition tosequences identified by SEQ ID NO or sequences related to these byvariations and by hybridization as described herein, other sequencesencoding unrelated (heterologous—that is, with insignificant sequencesimilarity to a Ferroportin1) polypeptides or peptides. These peptidesor polypeptides can be whole proteins, as occur naturally or as havebeen modified by design. Together, the nucleic acid sequences make upgenes for hybrid or fusion proteins. For example, an unrelated markersequence that facilitates purification (e.g., by affinity column) of thefused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence can be a hexa-histidine peptide, asprovided in the pQE vector (Qiagen, Inc.) and described in Gentz et al.,Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), or an HA tag (Wilson etal., Cell 37: 767 (1984)), or a sequence encoding glutathioneS-transferase of Schistosoma japonicum (vectors available fromPharmacia; see Smith, D. B. and Johnson K. S., Gene 67:31 (1988) andKaelin, W. G. et al., Cell 70:351 (1992)). For additional applications,the unrelated nucleic acid sequence can encode a peptide or polypeptidewhich is immunogenic or which enhances the immunogenicity of the fusionprotein or polypeptide. Nucleic acids of the invention also include, butare not limited to, nucleic acids comprising a structural gene and itsnaturally associated sequences that control gene expression.

The invention further relates to variants, including naturally-occurringallelic variants, of those nucleic acids described specifically hereinby DNA sequence, that encode variants of such polypeptides as thosehaving the amino acid sequences SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.Such variants include nucleic acids encoding variants of theabove-listed amino acid sequences, wherein those variants have several,such as 5 to 10, 1 to 5, or 3, 2 or 1 amino acids substituted, deleted,or added, in any combination. Variants include polynucleotides encodingpolypeptides with at least 95% but less than 100% amino acid sequenceidentity to the polypeptides described herein by amino acid sequence.Variant polynucleotides hybridize, under low to high stringencyconditions, to the alleles described specifically herein by DNAsequence. In one embodiment, variants have silent substitutions,additions and deletions that do not alter the properties and activitiesof the Ferroportin1. Allelic variants of the polynucleotides encodinghuman Ferroportin1 (SEQ ID NO:5), mouse Ferroportin1 (SEQ ID NO:3), andzebrafish Ferroportin1 (SEQ ID NO:1) will be identified as mapping tochromosomal locations corresponding to the chromosomal locations of thewild type genes.

Orthologous genes are gene loci in different species that aresufficiently similar to each other in their nucleotide sequences tosuggest that they originated from a common ancestral gene. Orthologousgenes arise when a lineage splits into two species, rather than when agene is duplicated within a genome. Proteins that are orthologs areencoded by genes of two different species, wherein the genes are said tobe orthologous.

The invention further relates to polynucleotides encoding polypeptideswhich are orthologous to those polypeptides having a specific amino acidsequence described herein, such as the amino acid sequences (SEQ IDNO:2), (SEQ ID NO:4), and (SEQ ID NO:6). These polynucleotides, whichcan be called ortholog polynucleotides, encode orthologous polypeptidesthat can range in amino acid sequence identity to a reference amino acidsequence described herein, from about 65% to less than 100%, butpreferably 70% to 80%, more preferably 80% to 90%, and still morepreferably 90% to less than 100%. Orthologous polypeptides can also bethose polypeptides that range in amino acid sequence similarity to areference amino acid sequence described herein from about 75% to 100%.The ortholog polynucleotides encode polypeptides that have similarfunctional characteristics (e.g., iron transport activity) and similartissue distribution, as appropriate to the organism from which theortholog polynucleotides can be isolated.

Ortholog polynucleotides can be isolated from (e.g., by cloning ornucleic acid amplification methods) a great number of species, as shownby the sample of Ferroportin1 proteins from evolutionarily divergentspecies described herein. Ortholog polynucleotides corresponding to SEQID NO:1, SEQ ID NO:3, and SEQ ID NO:5 are those which can be isolatedfrom mammals such as rat, dog, chimpanzee, monkey, baboon, pig, rabbitand guinea pig, for example.

Further variants that are fragments of the nucleic acids of theinvention may be used to synthesize full-length nucleic acids of theinvention, such as by use as primers in a polymerase chain reaction. Asused herein, the term primer refers to a single-stranded oligonucleotidewhich acts as a point of initiation of template-directed DNA synthesisunder appropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as DNA orRNA polymerase or reverse transcriptase) in an appropriate buffer and ata suitable temperature. The appropriate length of a primer depends onthe intended use of the primer, but typically ranges from 15 to 30nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the template,but must be sufficiently complementary to hybridize with a template. Theterm primer site refers to the area of the target DNA to which a primerhybridizes. The term primer pair refers to a set of primers including a5′ (upstream) primer that hybridizes with the 5′ end of the DNA sequenceto be amplified and a 3′ (downstream) primer that hybridizes with thecomplement of the 3′ end of the sequence to be amplified.

Further embodiments of the invention are nucleic acids that are at least80% identical over their entire length to a nucleic acid describedherein, for example a nucleic acid having the nucleotide sequence in SEQID NO:1, SEQ ID NO:3, and SEQ ID NO:5. Additional embodiments arenucleic acids, and the complements of such nucleic acids, having atleast 90% nucleotide sequence identity to the above-described sequences,and nucleic acids having at least 95% nucleotide sequence identity. Inpreferred embodiments, DNA of the present invention has 97% nucleotidesequence identity, 98% nucleotide sequence identity, or at least 99%nucleotide sequence identity with the DNA whose sequences are presentedherein.

Other embodiments of the invention are nucleic acids that are at least80% identical in nucleotide sequence to a nucleic acid encoding apolypeptide having an amino acid sequence as set forth in SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:6, and nucleic acids that are complementary tosuch nucleic acids. Specific embodiments are nucleic acids having atleast 90% nucleotide sequence identity to a nucleic acid encoding apolypeptide having an amino acid sequence as described in the listabove, nucleic acids having at least 95% sequence identity, and nucleicacids having at least 97% sequence identity. Also included in theinvention are nucleic acid molecules comprising at least 80%, 90%, 95%,or 97% of the coding region of any of SEQ ID NOs 1, 3 or 5.

The terms “complementary” or “complementarity” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. Complementarity between twosingle-stranded molecules may be “partial” in which only some of thenucleic acids bind, or it may be complete when total complementarityexists between the single-stranded molecules (that is, when A-T and G-Cbase pairing is 100% complete). The degree of complementarity betweennucleic acid strands has significant effects on the efficiency andstrength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, which depend onbinding between nucleic acid strands.

The invention further includes nucleic acids that hybridize to theabove-described nucleic acids, especially those nucleic acids thathybridize under stringent hybridization conditions. Preferred nucleicacid molecules meeting these hybridization criteria also encode apolypeptide having an iron transport function. “Stringent hybridizationconditions” or “high stringency conditions” generally occur within arange from about T_(m) minus 5° C. (5° C. below the strand dissociationtemperature or melting temperature (T_(m)) of the probe nucleic acidmolecule) to about 20° C. to 25° C. below T_(m). As will be understoodby those of skill in the art, the stringency of hybridization may bealtered in order to identify or detect molecules having identical orrelated polynucleotide sequences. An example of high stringencyhybridization follows. Hybridization solution is (6×SSC/10 mM EDTA/0.5%SDS/5× Denhardt's solution/100 μg/ml sheared and denatured salmon spermDNA). Hybridization is at 64-65° C. for 16 hours. The hybridized blot iswashed two times with 2×SSC/0.5% SDS solution at room temperature for 15minutes each, and two times with 0.2×SSC/0.5% SDS at 65° C., for onehour each. Further examples of high stringency conditions can be foundon pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds.,containing supplements up through Supplement 49, 2000). Examples ofhigh, medium, and low stringency conditions can be found on pages 36 and37 of WO 98/40404, which are incorporated herein by reference.

The invention further relates to nucleic acids obtainable by screeningan appropriate library with a probe having a nucleotide sequence such asthat set forth in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, or a probewhich consists of the coding region of any of these SEQ ID NOs, or aprobe which is a sufficiently long fragment of any of the above; andisolating the nucleic acid. Such probes generally can comprise at least15 nucleotides. Nucleic acids obtainable by such screenings may includeRNAs, cDNAs and genomic DNA, for example, encoding iron transportproteins of the Ferroportin1 protein family described herein.

Other nucleic acid embodiments are those comprising a nucleotidesequence encoding a contiguous portion of a polypeptide represented ashaving amino acid sequence SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6,wherein the portion is at least about 15 amino acids long, but canalternatively be at least 30 amino acids long or 60 amino acids long.The portion can be derived from amino acid sequence at the N-terminal,C-terminal or internal regions of SEQ ID NOs 2, 4 or 6.

Further uses for the nucleic acid molecules of the invention, whetherencoding a full-length Ferroportin1 protein or whether comprising acontiguous portion of a nucleic acid molecule such as one given in SEQID NO:1, 3 or 5, include use as markers for tissues in which the encodedprotein is preferentially expressed (to identify constitutivelyexpressed proteins or proteins produced at a particular stage of tissuedifferentiation or stage of development of a disease state); asmolecular weight markers on southern gels; as chromosome markers or tags(when labeled, for example with biotin, a radioactive label or afluorescent label) to identify chromosomes or to map related genepositions; to compare with endogenous DNA sequences in a mammal toidentify potential genetic disorders; as probes to hybridize and thusidentify, related DNA sequences; as a source of information to derivePCR primers for genetic fingerprinting; as a probe to “subtract-out”known sequences in the process of discovering other novel nucleic acidmolecules; for selecting and making oligomers for attachment to a “genechip” or other support, to be used, for example, for examination ofexpression patterns in embryonic development or in organs of an animalat a particular developmental stage.

Further methods to obtain nucleic acids encoding Ferroportin1 proteinsinclude PCR and variations thereof (e.g., “RACE” PCR and semi-specificPCR methods). Portions of the nucleic acids having a nucleotide sequenceset forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, (especially“flanking sequences” on either side of a coding region) can be used asprimers in methods using the polymerase chain reaction, to produce DNAfrom an appropriate template nucleic acid.

Once a fragment of the ferroportin1 gene is generated by PCR, it can besequenced, and the sequence of the product can be compared to other DNAsequences, for example, by using the BLAST Network Service at theNational Center for Biotechnology Information. The boundaries of theopen reading frame can then be identified using semi-specific PCR orother suitable methods such as library screening. Once the 5′ initiatormethionine codon and the 3′ stop codon have been identified, a PCRproduct encoding the full-length gene can be generated using cDNA as atemplate (the cDNA being generated from mRNA), with primerscomplementary to the extreme 5′ and 3′ ends of the gene or to theirflanking sequences. The full-length genes can then be cloned intoexpression vectors for the production of functional proteins.

In some embodiments of the invention, the nucleic acid molecules can bemodified at the base moiety, sugar moiety or phosphate backbone tochange the stability, hybridization or solubility properties of themolecules. For example, the deoxyribose phosphate backbone of thenucleic acids can be modified to generate peptide nucleic acids, or PNAs(Hyrup et al., Bioorganic and Medicinal Chemistry 4 :5-23, 1996). PNAsare nucleic acid mimics in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described by Hyrupet al. (1996) and Perry-O'Keefe et al., Proc. Natl. Acad. Sci. USA93:14670-14675, 1996. PNAs can be used in place of nucleic acids forsome applications, for example, as probes or primers for DNA sequenceanalysis and hybridization, or as antisense agents for sequence-specificmodulation of gene expression.

Nucleic acid molecules of the present invention can be incorporated intovarious constructs (e.g., plasmids, bacteriophages, viruses, artificialchromosomes) and incorporated into host cells in these constructs or inone or more chromosomes of the host cell, for example, for furthermanipulation of the sequences or for production of an encodedpolypeptide under suitable conditions for the growth or maintenance ofthe cells.

A host cell is a cell, or a descendant thereof, which has beentransfected by an exogenous DNA sequence using methods within the skillof those in the art. See, e.g., Graham et al. (1973) Virology 52:456,Sambrook et al. (1989) Molecular Cloning: a Laboratory Manual, ColdSpring Harbor Laboratories, New York, Davis et al. (1986) Basic Methodsin Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Moreparticularly, there are two major steps in transfection: first, theexogenous DNA must traverse the recipient (host) cell plasma membrane inorder to be exposed to the cell's transcription and replicationmachinery; and second, the DNA must either become stably integrated intothe host cell genome, or be capable of extra-chromosomal replication ata sufficient rate. A number of transfection methods have been describedin the art, such as calcium phosphate co-precipitation (Graham et al.(1973) Virol. 52:456-467), direct micro-injection into cultured cells(Capecchi, M. R. (1980) Cell 22:479-488), electroporation (Shigekawa etal. (1988) BioTechniques 6:742-751), liposome mediated gene transfer(Mannino et al. (1988) BioTechniques 6:682-690), lipid-mediatedtransfection (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

The invention also relates to isolated proteins or polypeptides such asthose encoded by nucleic acids of the present invention. Isolatedproteins can be purified from a natural source or can be maderecombinantly. Proteins or polypeptides referred to herein as “isolated”are proteins or polypeptides that exist in a state different from thestate in which they exist in cells in which they are normally expressedin an organism, and include proteins or polypeptides obtained by methodsdescribed herein, similar methods or other suitable methods, and alsoinclude essentially pure proteins or polypeptides, proteins orpolypeptides produced by chemical synthesis or by combinations ofbiological and chemical methods, and recombinant proteins orpolypeptides which are isolated. Thus, the term “isolated” as usedherein, indicates that the polypeptide in question exists in a physicalmilieu distinct from that in which it occurs in nature. Thus, “isolated”includes existing in membrane fragments and vesicles, membranefractions, liposomes, lipid bilayers and other artificial membranesystems. An isolated Ferroportin1 may be substantially isolated withrespect to the complex cellular milieu in which it naturally occurs, andmay even be purified essentially to homogeneity, for example asdetermined by PAGE or column chromatography (for example, HPLC), but mayalso have further cofactors or molecular stabilizers, such asdetergents, added to the purified protein to enhance activity. In oneembodiment, proteins or polypeptides are isolated to a state at leastabout 75% pure; more preferably at least about 85% pure, and still morepreferably at least about 95% pure, as determined by Coomassie bluestaining of proteins on SDS-polyacrylamide gels. Proteins orpolypeptides referred to herein as “recombinant” are proteins orpolypeptides produced by the expression of recombinant nucleic acids.

“Polypeptide” as used herein indicates a molecular chain of amino acidsand does not refer to a specific length of the product. Thus, peptides,oligopeptides and proteins are included within the definition ofpolypeptide. This term is also intended to include polypeptide that havebeen subjected to post-expression modifications such as, for example,glycosylations, acetylations, phosphorylations and the like.

In a preferred embodiment, an isolated polypeptide comprising aFerroportin1, a functional portion thereof, or a functional equivalentof the Ferroportin1, has at least one function characteristic of aFerroportin1, for example, transport activity, binding function (e.g., adomain which binds to a cofactor), or antigenic function (e.g., bindingof antibodies that also bind to a naturally-occurring Ferroportin1, asthat function is found in an antigenic determinant). Functionalequivalents can have activities that are quantitatively similar to,greater than, or less than, the reference protein. These proteinsinclude, for example, naturally occurring Ferroportin1 proteins that canbe purified from tissues in which they are produced (includingpolymorphic or allelic variants), variants (e.g., mutants) of thoseproteins and/or portions thereof. Such variants include mutantsdiffering by the addition, deletion or substitution of one or more aminoacid residues, or modified polypeptides in which one or more residuesare modified, and mutants comprising one or more modified residues. Theportions of the invention also include isolated polypeptides encoded bya nucleic acid molecule, wherein said nucleic acid molecule hybridizesto a complement of any of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 underhigh stringency conditions. Portions or fragments of a Ferroportin1 canrange in size from ten amino acid residues to the entire amino acidsequence minus one amino acid. An isolated polypeptide comprising afunctional portion of a Ferroportin1 can comprise at least 10 amino acidresidues of a cytoplasmic or extracellular domain of a Ferroportin1.

The isolated proteins of the invention preferably include mammalian irontransport proteins of the Ferroportin1 family of homologous proteins. Inpreferred embodiments, the extent of amino acid sequence identitybetween a polypeptide having one of the amino acid sequences SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:6, and the respective functionalequivalents of these polypeptides is at least about 80% or 88%. In otherembodiments, the degree of amino acid sequence identity between aFerroportin1 and its respective functional equivalent is at least about91%, at least about 94%, or at least about 97%.

The polypeptides of the invention also include those Ferroportin1proteins encoded by polynucleotides which are orthologous to thosepolynucleotides, the sequences of which are described herein in whole orin part. Ferroportin1 proteins which are orthologs to those describedherein by amino acid sequence, in whole or in part, are, for example,Ferroportin1 proteins of dog, rat, chimpanzee, monkey, rabbit, guineapig, baboon and pig, and are also embodiments of the invention.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimalcomparison. In the simplest concept of identity, two nucleic acidsequences or two amino acid sequences are compared after aligning themfor the maximum number of matches at the same position, without theintroduction of any gaps. In a somewhat more complex concept ofidentity, the sequences are aligned and gaps can be introduced in one orboth of a first and a second amino acid or nucleic acid sequence foroptimal alignment, and non-homologous (dissimilar) sequences can bedisregarded for comparison purposes. In a preferred embodiment, thelength of a reference sequence aligned for comparison purposes is atleast 30%, preferably at least 40%, more preferably at least 50%, evenmore preferably at least 60%, and even more preferably at least 70%,80%, or 90% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The invention also encompasses polypeptides having a lower degree ofidentity but having sufficient similarity in terms of structure andchemical characteristics so as to perform one or more of the samefunctions performed by the polypeptides described herein by amino acidsequence. Similarity for a polypeptide is determined by amino acidsubstitutions, which can be conservative amino acid substitutions. Forexample, the invention encompasses polypeptides with at least oneconservative amino acid substitution. Conservative substitutions arethose that replace a given amino acid residue in a polypeptide withanother amino acid residue of like characteristics. Conservativesubstitutions are likely to be phenotypically silent. Typically seen asconservative substitutions are the replacements, one for another, amongthe aliphatic amino acids Ala, Val, Leu, and Ile; interchange of thehydroxyl residues Ser and Thr, exchange of the acidic residues Asp andGlu, substitution between the amide residues Asn and Gln, exchange ofthe basic residues Lys and Arg and replacements among the aromaticresidues Phe, Tyr and Trp. Guidance concerning which amino acid changesare likely to be phenotypically silent is found in Bowie et al., Science247:1306-1310 (1990). TABLE Conservative Amino Acid SubstitutionsAromatic Phenylalanine Tryptophan Tyrosine Hydrophobic LeucineIsoleucine Valine Polar Glutamine Asparagine Basic Arginine LysineHistidine Acidic Aspartic Acid Glutamic Acid Small Alanine SerineThreonine Methionine Glycine

The comparison of sequences and determination of percent similaritybetween two sequences can be accomplished using a mathematicalalgorithm. (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereaux, J., eds., M. Stockton Press, NewYork, 1991). In a preferred embodiment, the percent similarity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the Wisconsin Package Version 10.0,Genetics Computer Group (GCG), Madison Wis., using, for example, aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent similarity between two nucleotide sequences isdetermined using the GAP program in the Wisconsin Package (Devereux, J.,et al., Nucleic Acids Res. 12(1):387 (1984)), using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of1, 2, 3, 4, 5, or 6. In another embodiment, the percent similaritybetween two amino acid or nucleotide sequences is determined using thealgorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which hasbeen incorporated into the ALIGN program (version 2.0), using a PAM120weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acids and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the BLASTN, BLASTP,BLASTX, TBLASTN, TBLASTX programs (version 2.0) or PSI-BLAST 2.1programs based on Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)).BLAST nucleotide searches can be performed with the BLASTN program, forexample, with default parameters matrix=BIOSUM62, gap existence cost=11,per residue gap cost=1, lambda ratio=0.85, filtered, to obtainnucleotide sequences homologous to (with calculatably significantsimilarity to) the nucleic acid molecules of the invention. BLASTprotein searches can be performed with the BLASTP program, for example,with default parameters scoring matrix=BIOSUM62, word size=3, Evalue=10, gap costs=11, 1 and alignments=50, to obtain amino acidsequences homologous to the proteins of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (Nucleic Acids Res. 25(17):3389-3402(1997)). When utilizing BLAST and gapped BLAST programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused.

Within the invention are isolated nucleic acid molecules having at least80%, 85%, 90%, 95% and 97% sequence similarity to a nucleic acidencoding a polypeptide comprising the amino acid sequence SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:6. Also within the invention are isolatednucleic acid molecules which hybridize under high stringency conditionsto nucleic acid consisting of the coding regions of SEQ ID NO:1, SEQ IDNO:3 or SEQ ID NO:5.

The invention further relates to fusion proteins, comprising aFerroportin1 or functional portion thereof (as described above) as afirst moiety, linked to second moiety or to multiple moieties notoccurring in the Ferroportin1 as found in nature. Thus, a second moietycan be an amino acid, peptide or polypeptide. The second moiety can bein an N-terminal location, C-terminal location or internal to the fusionprotein, or multiple heterologous moieties can be in multiple locations.In one embodiment, the fusion protein comprises a Ferroportin1 orportion thereof having iron transport function as the first moiety, anda second moiety comprising a linker sequence and an affinity ligand.Fusion proteins can be produced by a variety of methods. For example, afusion protein can be produced by the insertion of a ferroportin1 geneor portion thereof into a suitable expression vector, such as BluescriptSK+/−(Stratagene), pGEX-4T-2 (Pharmacia), pET-24(+) (Novagen), orvectors of similar construction. The resulting construct can beintroduced into a suitable host cell for expression. Upon expression,fusion protein can be purified from cells by means of a suitableaffinity matrix (See e.g., Current Protocols in Molecular Biology,Ausubel, F. M. et al., eds., Vol. 2, pp. 16.4.1-16.7.8, containingsupplements up through Supplement 49, 2000).

The invention also relates to enzymatically produced, syntheticallyproduced, or recombinantly produced portions of a Ferroportin1 protein.Portions of a Ferroportin1 can be made which have full or partialfunction on their own, or which when mixed together (though fully,partially, or nonfunctional alone), spontaneously assemble with one ormore other polypeptides to reconstitute a functional protein having atleast one function characteristic of a Ferroportin1.

Fragments of a Ferroportin1 can be produced by direct peptide synthesis,for example those using solid-phase techniques (Roberge, J. Y. et al.,Science 269:202-204 (1995); Merrifield, J., J. Am. Chem. Soc.85:2149-2154 (1963)). Peptide or polypeptide synthesis can be performedusing manual techniques or by automation. Automated synthesis can becarried out using, for instance, an Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Various fragments of a Ferroportin1 can besynthesized separately and combined using chemical methods.

One aspect of the invention is a peptide or polypeptide having the aminoacid sequence of a portion of a Ferroportin1 protein which ishydrophilic rather than hydrophobic, and ordinarily can be detected asfacing the outside of the cell membrane. Such a peptide or polypeptidecan be thought of as being an extracellular domain of the Ferroportin1,or a mimetic of said extracellular domain. Peptides or polypeptidescomprising at least 10 amino acid residues of a cytoplasmic orextracellular domain of human, mouse or zebrafish Ferroportin1 can besynthesized.

The term “mimetic” as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of theFerroportin1 of interest, or one or more portions thereof, and, as such,is able to effect some or all of the functions of a Ferroportin1.

Portions of a Ferroportin1 can be prepared by enzymatic cleavage of theisolated protein, or can be made by chemical synthesis methods. Portionsof a Ferroportin1 can also be made by recombinant DNA methods in whichrestriction fragments, or fragments that may have undergone furtherenzymatic processing, or synthetically made DNAs are joined together toconstruct an altered ferroportin1 gene. The gene can be made such thatit encodes one or more desired portions of a Ferroportin1. Theseportions of Ferroportin1 can be entirely homologous to a knownFerroportin1, or can be altered in amino acid sequence relative tonaturally occurring Ferroportin1 proteins to enhance or introducedesired properties such as solubility, stability, or affinity to aligand. A further feature of the gene can be a sequence encoding anN-terminal signal peptide directed to the plasma membrane.

Another aspect of the invention relates to a method of producing aFerroportin1 protein, variants or portions thereof, and to expressionsystems and host cells containing a vector appropriate for expression ofa Ferroportin1 protein.

Cells that express a Ferroportin1, a variant or a portion thereof, or anortholog of a Ferroportin1 described herein by amino acid sequence, canbe made and maintained in culture, under conditions suitable forexpression, to produce protein in the cells for cell-based assays, or toproduce protein for isolation. These cells can be procaryotic oreucaryotic. Examples of procaryotic cells that can be used forexpression include Escherichia coli, Salmonella typhimurium and Bacillussubtilis. Examples of eucaryotic cells that can be used for expressioninclude yeasts such as Saccharomyces cerevisiae, Schizosaccharomycespombe, Pichia pastoris and other lower eucaryotic cells, and cells ofhigher eucaryotes such as those from insects and mammals, such asprimary cells and cell lines such as CHO, HeLa, 3T3, BHK, COS, humankidney 293 and Jurkat cells. (See, e.g., Ausubel, F. M. et al., eds.Current Protocols in Molecular Biology, Greene Publishing Associates andJohn Wiley & Sons, Inc., containing Supplements up through Supplement49, 2000)).

In one embodiment, host cells that produce a recombinant Ferroportin1,or a portion thereof, a variant, or an ortholog of a Ferroportin1described herein by amino acid sequence, can be made as follows. A geneencoding a Ferroportin1, variant or a portion thereof can be insertedinto a nucleic acid vector, e.g., a DNA vector, such as a plasmid,phage, cosmid, phagemid, virus, virus-derived vector (e.g., SV40,vaccinia, adenovirus, fowl pox virus, pseudorabies viruses,retroviruses) or other suitable replicon, which can be present in asingle copy or multiple copies, or the gene can be integrated in a hostcell chromosome. A suitable replicon or integrated gene can contain allor part of the coding sequence for a Ferroportin1 or variant, operablylinked to one or more expression control regions whereby the codingsequence is under the control of transcription signals and linked toappropriate translation signals to permit translation. The vector can beintroduced into cells by a method appropriate to the type of host cells(e.g., transfection, electroporation, infection). For expression fromthe Ferroportin1 gene, the host cells can be maintained underappropriate conditions (e.g., in the presence of inducer, normal growthconditions, etc.). Proteins or polypeptides thus produced can berecovered (e.g., from the cells, as in a membrane fraction, from theperiplasmic space of bacteria, from culture medium) using suitabletechniques. Appropriate membrane targeting signal peptides may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signalpeptides that do no naturally occur with a Ferroportin1.

Polypeptides of the invention can be recovered and purified from cellcultures (or from their primary cell source) by well-known methodsincluding ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and high performanceliquid chromatography. Known methods for refolding protein can be usedto regenerate active conformation if the polypeptide is denatured duringisolation or purification.

The host cells of the invention can be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichFerroportin1 coding sequences have been introduced. Such host cells canthen be used to create non-human transgenic animals in which exogenousferroportin1 sequences have been introduced into their genome, orhomologous recombinant animals in which endogenous ferroportin1sequences have been altered. Such animals are useful for studying thefunction and/or activity of Ferroportin1, and for identifying and/orevaluating modulators of Ferroportin1 activity. As used herein, a“transgenic animal” is a non-human animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. “Exogenous” as used in the context ofa transgenic animal, means different from that of the unalteredrecipient host cell. As used herein, a “homologous recombinant animal”is a non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous weh gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingFerroportin1 encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The ferroportin1 cDNA sequence can be introduced as a transgene into thegenome of a non-human animal. Alternatively, a nonhuman homolog of thehuman ferroportin1 gene can be isolated based on hybridization to thehuman or mouse ferroportin1 cDNA and used as a transgene. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene. Oneor more tissue-specific regulatory sequences can be operably linked tothe ferroportin1 transgene to direct expression of Ferroportin1 proteinto particular cells. Methods for generating transgenic animals viaembryo manipulation and microinjection, particularly animals such asmice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No.4,873,191 and in Hogan, Manipulating the Mouse Embryo (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similarmethods are used for production of other transgenic animals. Atransgenic founder animal can be identified based upon the presence ofthe ferroportin1 transgene in its genome and/or expression offerroportin1 mRNA in tissues or cells of the animals. A transgenicfounder animal can then be used to breed additional animals carrying thetransgene. Transgenic animals carrying a transgene encoding Ferroportin1can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a ferroportin1 gene (e.g., a human or anon-human homolog of the gene encoding Ferroportin1, e.g., a murineferroportin1 gene) into which a deletion, addition or substitution hasbeen introduced to thereby alter, e.g., functionally disrupt, theferroportin1 gene. In a preferred embodiment, the vector is designedsuch that, upon homologous recombination, the endogenous ferroportin1gene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector).

Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous ferroportin1 gene is mutated or otherwisealtered but still encodes functional protein (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous Ferroportin1 protein). In the homologous recombinationvector, the altered portion of the ferroportin1 gene is flanked at its5′ and 3′ ends by additional nucleic acid of the ferroportin1 gene toallow for homologous recombination to occur between the exogenousferroportin1 gene carried by the vector and an endogenous ferroportin1gene in an embryonic stem cell. The additional flanking ferroportin1nucleic acid is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the vector(see, e.g., Thomas and Capecchi (1987) Cell 51:503 for a description ofhomologous recombination vectors). The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced ferroportin1 gene has homologously recombined with theendogenous ferroportin1 gene are selected (see., e.g., Li et al. (1992)Cell 69:915). The selected cells are then injected into a blastocyst ofan animal (e.g., a mouse) to form aggregation chimeras (see, e.g.,Bradley in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimericembryo can then be implanted into a suitable pseudopregnant femalefoster animal and the embryo brought to term. Progeny harboring thehomologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley (1991) Current Opinion inBio/Technology 2:823-829 and in WO 90/11354, WO 91/01140, WO 92/0968,and WO 93/04169.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355). If acre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and WO 97/07668 and WO 97/07669. In brief, a cell,e.g., a somatic cell, from the transgenic animal can be isolated andinduced to exit the growth cycle and enter G₀ phase. The quiescent cellcan then be fused, e.g., through the use of electrical pulses, to anenucleated oocyte from an animal of the same species from which thequiescent cell is isolated. The reconstructed oocyte is then culturedsuch that it develops to morula or blastocyte and then transferred topseudopregnant female foster animal. The offspring borne of this femalefoster animal will be a clone of the animal from which the cell, E.g.,the somatic cell, is isolated.

In a further aspect of the invention are methods for assessing thetransport function of any of the Ferroportin1 proteins or polypeptidesdescribed herein, including orthologs, and in variations of these,methods for identifying an inhibitor (or an enhancer) of such functionand methods for assessing the transport function in the presence of acandidate inhibitor or a known inhibitor.

A variety of systems comprising living cells can be used for thesemethods. Cells to be used in iron transport assays, and further inmethods for identifying an inhibitor or enhancer of this function,express one or more Ferroportin1 proteins. Cells for use in cell-basedassays described herein can be drawn from a variety of sources, such asisolated primary cells of various organs and tissues wherein aFerroportin1 protein is naturally expressed. In some cases, the cellscan be from adult organs, and in some cases, from embryonic or fetalstructures, such as placenta, yolk sac, heart, lung, liver, intestine,skeletal muscle, kidney and the like. Cells for this purpose can alsoinclude cells cultured as fragments of organs or in conditionssimulating the cell type and/or tissue organization of organs, in whichartificial materials may be used as substrates for cell growth. Othertypes of cells suitable for this purpose include cells of a cell strainor cell line (ordinarily comprising cells considered to be“transformed”) transfected to express one or more types of Ferroportin1.

A further embodiment of the invention is a method for detecting, in asample of cells, a Ferroportin1 protein, a portion or fragment thereof,a fusion protein comprising a Ferroportin1 or a portion thereof, or anortholog as described herein, wherein the cells can be, for instance,cells of a tissue, primary culture cells, or cells of a cell line,including cells into which nucleic acid has been introduced. The methodcomprises adding to the sample an agent that specifically binds to theprotein, and detecting the agent specifically bound to the protein.Appropriate washing steps can be added to reduce nonspecific binding tothe agent. The agent can be, for example, an antibody, a ligand or asubstrate or cofactor mimic. The agent can have incorporated into it, orhave bound to it, covalently or by high affinity non-covalentinteractions, for instance, a label that facilitates detection of theagent to which it is bound, wherein the label can be, but is not limitedto, a phosphorescent label, a fluorescent label, a biotin or avidinlabel, or a radioactive label. The means of detection of a Ferroportin1can vary, as appropriate to the agent and label used. For example, foran antibody that binds to the Ferroportin1, the means of detection maycall for binding a second antibody, which has been conjugated to anenzyme, to the antibody which binds the Ferroportin1, and detecting thepresence of the second antibody by means of the enzymatic activity ofthe conjugated enzyme.

Similar principles can also be applied to a cell lysate, membranefraction, or a more purified preparation of proteins from cells that maycomprise a Ferroportin1 protein of interest, for example in the methodsof immunoprecipitation, immunoblotting, immunoaffinity methods, that inaddition to detection of the particular Ferroportin1, can also be usedin purification steps, and qualitative and quantitative immunoassays.See, for instance, chapters 11 through 14 in Antibodies: A LaboratoryManual, E. Harlow and D. Lane, eds., Cold Spring Harbor Laboratory,1988.

Isolated Ferroportin1 protein or, an antigenically similar portionthereof, especially a portion that is soluble (e.g., a peptide or afusion polypeptide comprising at least 10 contiguous amino acid residuesof a Ferroportin1), can be used in a method to select and identifymolecules which bind specifically to the Ferroportin1. Fusion proteinscomprising all of, or a portion of, the Ferroportin1 linked to a secondmoiety not occurring in the Ferroportin1 as found in nature, can beprepared for use in another embodiment of the method. Suitable fusionproteins for this purpose include those in which the second moietycomprises an affinity ligand (e.g., an enzyme, antigen, epitope).Ferroportin1 fusion proteins can be produced by the insertion of a geneencoding the Ferroportin1 or a variant thereof, or a suitable portion ofsuch gene into a suitable expression vector which encodes an affinityligand (e.g., pGEX-4T-2 and pET-15b, encoding glutathione S-transferaseand His-Tag affinity ligands, respectively). The expression vector canbe introduced into a suitable host cell for expression. Host cells arelysed and the lysate, containing fusion protein, can be bound to asuitable affinity matrix by contacting the lysate with an affinitymatrix.

In one embodiment, the fusion protein can be immobilized on a suitableaffinity matrix under conditions sufficient to bind the affinity ligandportion of the fusion protein to the matrix, and is contacted with oneor more candidate binding agents (e.g., a mixture of peptides orcompounds of a library) to be tested, under conditions suitable forbinding of the binding agents to the Ferroportin1 portion of the boundfusion protein. Next, the affinity matrix with bound fusion protein canbe washed with a suitable wash buffer to remove unbound candidatebinding agents and non-specifically bound candidate binding agents.Those agents which remain bound can be released by contacting theaffinity matrix with fusion protein bound thereto with a suitableelution buffer. Wash buffer can be formulated to permit binding of thefusion protein to the affinity matrix, without significantly disruptingbinding of specifically bound binding agents. In this aspect, elutionbuffer can be formulated to permit retention of the fusion protein bythe affinity matrix, but can be formulated to interfere with binding ofthe candidate binding agents to the target portion of the fusionprotein. For example, a change in the ionic strength or pH of theelution buffer can lead to release of specifically bound agent, or theelution buffer can comprise a release component or components designedto disrupt binding of specifically bound agent to the target portion ofthe fusion protein.

Immobilization can be performed prior to, simultaneous with, or after,contacting the fusion protein with candidate binding agent, asappropriate. Various permutations of the method are possible, dependingupon factors such as the candidate molecules tested, the affinitymatrix-ligand pair selected, and elution buffer formulation. Forexample, after the wash step, fusion protein with binding agentmolecules bound thereto can be eluted from the affinity matrix with asuitable elution buffer (a matrix elution buffer, such as glutathionefor a GST fusion). Where the fusion protein comprises a cleavablelinker, such as a thrombin cleavage site, cleavage from the affinityligand can release a portion of the fusion with the candidate agentbound thereto. Bound agent molecules can then be released from thefusion protein or its cleavage product by an appropriate method, such asextraction.

One or more candidate binding agents can be tested simultaneously. Wherea mixture of candidate binding agents is tested, those found to bind bythe foregoing processes can be separated (as appropriate) and identifiedby suitable methods (e.g., PCR, sequencing, chromatography). Largelibraries of candidate binding agents produced by combinatorial chemicalsynthesis or by other methods can be tested (see e.g., Ohlmeyer, M. H.J. et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt,S. H. et al., Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993), relatingto tagged compounds; see also Rutter, W. J. et al. U.S. Pat. No.5,010,175; Huebner, V. D. et al., U.S. Pat. No. 5,182,366; and Geysen,H. M., U.S. Pat. No. 4,833,092). Where binding agents selected from acombinatorial library by the present method carry unique tags,identification of individual biomolecules by chromatographic methods ispossible. Where binding agents do not carry tags, chromatographicseparation, followed by mass spectrometry to ascertain structure, can beused to identify binding agents selected by the method, for example.

The invention also comprises a method for identifying an agent whichinhibits interaction between a Ferroportin1 protein (e.g., onecomprising the amino acid sequence in SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:6), and a ligand of said protein. The Ferroportin1 can be onedescribed by amino acid sequence herein, a portion or fragment thereof,a variant thereof, or an ortholog thereof, or a Ferroportin1 fusionprotein. Here, a ligand can be, for instance, a substrate (e.g., Fe²⁺),or a substrate mimic, an antibody, or a compound, such as a smallmolecule or peptide, that binds with specificity to a site on theprotein. The method comprises combining, not limited to a particularorder, the Ferroportin1 protein, the ligand of the protein, and acandidate agent to be assessed for its ability to inhibit interactionbetween the protein and the ligand, under conditions appropriate forinteraction between the protein and the ligand (e.g., pH, salt,temperature conditions conducive to appropriate conformation andmolecular interactions); determining the extent to which the protein andligand interact; and comparing (1) the extent of protein-ligandinteraction in the presence of candidate agent with (2) the extent ofprotein-ligand interaction in the absence of candidate agent, wherein if(1) is less than (2), then the candidate agent is one which inhibitsinteraction between the protein and the ligand.

The method can be facilitated, for example, by using an experimentalsystem which employs a solid support (column chromatography matrix, wallof a plate, microtiter wells, column pore glass, pins to be submerged ina solution, beads, etc.) to which the protein can be attached.Accordingly, in one embodiment, the protein can be fixed to a solidphase directly or indirectly, by a linker. The candidate agent to betested is added under conditions conducive for interaction and bindingto the protein. The ligand is added to the solid phase system underconditions appropriate for binding. Excess ligand is removed, as by aseries of washes done under conditions that do not disruptprotein-ligand interactions. Detection of bound ligand can befacilitated by using a ligand that carries a label (e.g., fluorescent,chemiluminescent, radioactive). In a control experiment, protein andligand are allowed to interact in the absence of any candidate agent,under conditions otherwise identical to those used for the “test”conditions where candidate inhibiting agent is present, and any washesused in the test conditions are also used in the control. The extent towhich ligand binds to the protein in the presence of candidate agent iscompared to the extent to which ligand binds to the protein in theabsence of the candidate agent. If the extent to which interaction ofthe protein and the ligand occurs is less in the presence of thecandidate agent than in the absence of the candidate agent, thecandidate agent is an agent which inhibits interaction between theprotein and the ligand of the protein.

In a further embodiment, an inhibitor (or an enhancer) of a Ferroportin1protein can be identified. The method comprises steps which are, or arevariations of, the following: contacting the cells with Fe²⁺ underconditions allowing uptake of the Fe²⁺, wherein the Fe²⁺ can be labeledfor convenience of detection; washing away extracellular Fe²⁺,contacting a first aliquot of the cells with an agent being tested as aninhibitor (or enhancer) of iron export, while maintaining a secondaliquot of cells under the same conditions but without contact with theagent; and determining (e.g., by a quantitative measurement) ironexported from the first and second aliquots of cells; wherein a lesserquantity of iron in the first aliquot compared to that in the secondaliquot is indicative that the agent is an inhibitor of iron export by aFerroportin1 protein. A greater quantity of extracellular iron found inthe first aliquot compared to that in the second aliquot is indicativethat the agent is an enhancer of iron export by a Ferroportin1 protein.

A particular embodiment of identifying an inhibitor or enhancer of ironexport function employs the above steps, but also employs additionalsteps preceding those given above: introducing into cells of a cellstrain or cell line (“host cells” for the intended introduction of, orafter the introduction of, a vector) one or more vectors or RNAscomprising a ferroportin1 gene, wherein expression of the gene can beregulatable or constitutive, and providing conditions to the host cellsunder which expression of the gene can occur, and under which iron canbe taken up by the host cells.

The terms “contacting” and “combining” as used herein in the context ofbringing molecules into close proximity to each other, can beaccomplished by conventional means. For example, when referring tomolecules that are soluble, contacting is achieved by adding themolecules together in a solution. “Contacting” can also be adding anagent to a test system, such as a vessel containing cells in tissueculture.

The term “inhibitor” or “antagonist”, as used herein, refers to an agentwhich blocks, diminishes, inhibits, hinders, limits, decreases, reduces,restricts or interferes with iron export from a cell, or alternativelyor additionally, prevents or impedes the cellular effects associatedwith iron export. The term “enhancer” or “agonist”, as used herein,refers to an agent which augments, enhances, or increases iron exportfrom a cell.

In order to produce a “host cell” type suitable for iron uptake assaysand for assays derived therefrom for identifying inhibitors or enhancersthereof, a nucleic acid vector can be constructed to comprise a geneencoding an iron transport protein, for example, human Ferroportin1, amutant or variant thereof, an ortholog of the human protein, such asporcine or bovine orthologs or orthologs found in other mammals, or aFerroportin1 family protein of origin in an organism other than amammal. The gene of the vector can be regulatable, such as by theplacement of the gene under the control of an inducible or repressiblepromoter in the vector (e.g., inducible or repressible by a change ingrowth conditions of the host cell harboring the vector, such asaddition of inducer, binding or functional removal of repressor from thecell millieu, or change in temperature) such that expression of theferroportin1 gene can be turned on or initiated by causing a change ingrowth conditions, thereby causing the protein encoded by the gene to beproduced, in host cells comprising the vector, as a plasma membraneprotein. Alternatively, the ferroportin1 gene can be constitutivelyexpressed.

A vector comprising a ferroportin1 gene, such as a vector describedherein, can be introduced into host cells by a means appropriate to thevector and to the host cell type. For example, commonly used methodssuch as electroporation, transfection, for instance, transfection usingCaCl₂, and transduction (as for a virus or bacteriophage) can be used.Host cells can be, for example, mammalian cells such as primary culturecells or cells of cell lines such as COS cells, 293 cells or Jurkatcells. Host cells can also be, in some cases, cells derived frominsects, cells of insect cell lines, bacterial cells, such as E. coli,or yeast cells, such as S. cerevisiae. It is preferred that the ironexport protein whose function is to be assessed, with or without acandidate inhibitor or enhancer, be produced in host cells whoseancestor cells originated in a species related to the species of originof the ferroportin1 gene encoding the Ferroportin1 protein. For example,it is preferable that tests of function or of inhibition or enhancementof a mammalian Ferroportin1 be carried out in host mammalian cellsproducing the Ferroportin1, rather than in bacterial cells or yeastcells.

Host cells comprising a vector comprising a regulatable ferroportin1gene can be treated so as to allow expression of the ferroportin1 geneand production of the encoded protein (e.g., by contacting the cellswith an inducer compound that effects transcription from an induciblepromoter operably linked to the ferroportin1 gene).

The test agent (e.g., an agonist or antagonist) is added to the cells tobe used in an iron export assay, under conditions suitable forproduction and/or maintenance of the expressed Ferroportin1 in aconformation appropriate for association of the Ferroportin1 with testagent and substrate. For example, conditions under which an agent isassessed, such as media and temperature requirements, can initially besimilar to those necessary for transport of iron substrate across theplasma membrane. One of ordinary skill in the art will know how to varyexperimental conditions depending upon the biochemical nature of thetest agent. The test agent can be added to the cells before or after theaddition of an iron substrate. The concentration at which the test agentcan be evaluated can be varied, as appropriate, to test for an increasedeffect with increasing concentrations.

Test agents to be assessed for their effects on iron transport can beany chemical (element, molecule, compound), made synthetically, made byrecombinant techniques or isolated from a natural source. For example,test agents can be peptides, polypeptides, peptoids, sugars, hormones,or nucleic acid molecules, such as antisense nucleic acid molecules. Inaddition, test agents can be small molecules or molecules of greatercomplexity made by combinatorial chemistry, for example, and compiledinto libraries. These libraries can comprise, for example, alcohols,alkyl halides, amines, amides, esters, aldehydes, ethers and otherclasses of organic compounds. Test agents can also be natural orgenetically engineered products isolated from lysates of cells,bacterial, animal or plant, or can be the cell lysates themselves.Presentation of test compounds to the test system can be in either anisolated form or as mixtures of compounds, especially in initialscreening steps.

Thus, the invention relates to a method for identifying agents whichalter iron export, the method comprising providing the test agent to thecell (wherein “cell” includes the plural, and can include cells of acell strain, cell line or culture of primary cells or organ culture, forexample), under conditions suitable for binding to its target, whetherto the Ferroportin1 itself or to another target on or in the cell,wherein the cell comprises a Ferroportin1.

The cells to be tested for the effect of an agent on iron export can be“loaded” with iron by incubation of the cells with iron under conditionsappropriate for iron uptake. The cells can be, for example, cells oftransformed cell lines such as HeLa or 293 cells, fibroblasts,transformed fibroblasts or oocytes of Xenopus laevis or anotherappropriate species. The cells can also be cells transfected withnucleic acid encoding Ferroportin1, such that the cell expresses theFerroportin1 protein to be tested for the effect of an agent. The ironcan be labeled to facilitate its detection, for example, with aradioactive isotope.

The cells so loaded with iron are then washed with buffer or mediumsufficient to remove iron external to the cells. The cells can then bedivided into two equal aliquots, or two aliquots of known cell numbers.To one aliquot is added the agent to be tested for its effect on irontransport. To the other aliquot is added a volume of buffer, medium,etc. equivalent to that in which the agent added to the first aliquotwas dissolved. The two aliquots of cells are then kept under the sameculture conditions for a period of time to allow for the export of iron.After this period, the cells of each aliquot are separated from theirsurrounding medium, for example by centrifugation, and, for isolation ofthe cell pellet, by one or more additional washing steps. The medium canbe collected in each case, and aliquots of each can be assayed forexported iron. Where the iron is radioactively labeled, the medium canbe tested for radioactivity, as by scintillation counting.Alternatively, the cells or aliquots of the cells can be collected afterthe period of time allowing for iron export, and the cells can be lysedto prepare a cell extract to be assayed for iron retained in the cells.Where the cells to which an agent was added retain more iron than thecontrol cells not receiving agent, the agent is an inhibitor of ironexport. Where the cells to which an agent was added retain less ironthan the control cells, the agent is an enhancer of iron export. If thecell medium is assayed, where the cells receiving agent export less ironinto the medium than the control cells, then the agent is an inhibitorof iron transport. If the cells receiving agent export more iron intothe medium than the control cells, then the agent is an enhancer of irontransport.

An agent determined to be an inhibitor (or enhancer) of Ferroportin1function, such as iron binding and/or iron export, can be administeredto cells in culture, or in vivo, to a mammal (e.g. human) to inhibit (orenhance) Ferroportin1 function. Such an agent may be one that actsdirectly on the Ferroportin1 protein (for example, by binding) or canact on an intermediate in a biosynthetic pathway to produceFerroportin1, such as transcription of the ferroportin1 gene, processingof the mRNA, or translation of the mRNA. An example of such an agent isantisense oligonucleotide.

Cell-free assays can also be used to measure the transport of ironacross a membrane, and therefor also to assess a test treatment or testagent for its effect on the rate or extent of iron transport. IsolatedFerroportin1, for example in the presence of a detergent that preservesthe native 3-dimensional structure of the Ferroportin1 protein, orpartially purified Ferroportin1 protein, can be used in an artificialmembrane system typically used to preserve the native conformation andactivity of membrane proteins. Such systems include liposomes,artificial bilayers of phospholipids, isolated plasma membrane such ascell membrane fragments, cell membrane fractions, or cell membranevesicles, and other systems in which the Ferroportin1 protein can beproperly oriented within the membrane to have transport activity. Assaysfor transport activity can be performed using methods analogous to thosethat can be used in cells expressing a Ferroportin1 protein whosefunction is to be measured. A labeled (e.g., radioactively labeled) ironsubstrate can be incubated on one side of a bilayer or in a suspensionof liposomes constructed to integrate a properly oriented Ferroportin1protein. The accumulation of iron with time can be measured, usingappropriate means to detect the label (e.g., scintillation counting ofmedium on each side of the bilayer, or of the contents of liposomesversus the surrounding medium). Assays such as these can be adapted touse for the testing of agents which might interact with the Ferroportin1to produce an inhibitory or an enhancing effect on the rate or extent ofiron transport. That is, the above-described assay can be done in thepresence or absence of the agent to be tested, and the results compared.

For examples of isolation of membrane proteins (ADP/ATP carrier anduncoupling protein), reconstitution into phospholipid vesicles, andassays of transport, see Klingenberg, M. et al., Methods Enzymol.260:369-389 (1995). For an example of a membrane protein (phosphatecarrier of Saccharomyces cerevisiae) that was purified and solubilizedfrom E. coli inclusion bodies, see Schroer, A. et al., J. Biol. Chem.273:14269-14276 (1998). The Glut1 glucose transporter of rat has beenexpressed in yeast. A crude membrane fraction of the yeast was preparedand reconstituted with soybean phospholipids into liposomes. Glucosetransport activity could be measured in the liposomes (Kasahara, T. andKasahara, M., J. Biol. Chem. 273:29113-29117 (1998)). Similar methodscan be applied to the proteins and polypeptides of the invention.

Another embodiment of the invention is a method for inhibiting ironexport in Ferroportin1-expressing cells of a mammal (e.g., a human),comprising administering to the mammal a therapeutically effectiveamount of an inhibitor of the transport function of Ferroportin1,thereby decreasing iron in the circulation. Hemochromatosis can be dueto the inheritance of a mutant gene or due to secondary iron overloadfrom an iron-loading anemia such as thalassemia or sideroblastic anemia.Where it is desirable to reduce the uptake of iron into the circulatorysystem through the intestine, for example, in the treatment ofhemochromatosis in a human, one or more inhibitors of Ferroportin1 canbe administered in an effective dose, and by an effective route, forexample, orally, or by an indwelling device that can deliver doses tothe small intestine. The inhibitor can be one identified by methodsdescribed herein, or can be one that is, for instance, structurallyrelated to an inhibitor identified by methods described herein (e.g.,having chemical adducts to better stabilize or solubilize theinhibitor). The invention further relates to compositions comprisinginhibitors of iron uptake in a mammal, which may further comprisepharmaceutical carriers suitable for administration to a subject mammal,such as sterile solubilizing or emulsifying agents.

A further embodiment of the present invention is a method of enhancingor increasing iron uptake into the body, such as enhancing or increasingiron uptake in the small intestine (e.g., to treat a malabsorptionsyndrome or anemia). In this embodiment, a therapeutically effectiveamount of an enhancer of the transport function of Ferroportin1 can beadministered to a mammalian subject, with the result that iron uptake inthe small intestine is enhanced. In this embodiment, one or moreenhancers of a Ferroportin1 protein is administered in an effective doseand by a route (e.g., orally or by a device, such as an indwellingcatheter or other device) which can deliver doses to the gut. Theenhancer of Ferroportin1 function can be identified by methods describedherein or can be one that is structurally similar to an enhanceridentified by methods described herein.

The invention further relates to antibodies that bind to an isolated orrecombinant Ferroportin1, including portions of antibodies, which canspecifically recognize and bind to one or more Ferroportin1 proteins.The antibodies and portions thereof of the invention include those whichbind to Ferroportin1 proteins of zebrafish, or Ferroportin1 proteins ofmouse or other mammalian species. In a specific embodiment, theantibodies bind to a naturally occurring Ferroportin1 of humans. Theantibodies can be used in various methods to detect or to purify aprotein of the present invention or a portion thereof such as ELISA,western blotting or immunoaffinity chromatography, to inhibit thefunction of a protein in a method of therapy, or to selectivelyinactivate an active site, or to study other aspects of the structure ofthese proteins, for example.

The antibodies of the present invention can be polyclonal or monoclonal.The term antibody is intended to encompass both polyclonal andmonoclonal antibodies. Antibodies of the present invention can be raisedagainst an appropriate immunogen, including proteins or polypeptides ofthe present invention, such as an isolated or recombinant Ferroportin1or portions thereof, or synthetic molecules, such as synthetic peptides(e.g., conjugated to a suitable carrier). Preferred embodiments areantibodies that bind to any of the following, and which may cross-reactwith Ferroportin1 proteins of several species: zebrafish Ferroportin1,mouse Ferroportin1, or human Ferroportin1. The immunogen can be apolypeptide comprising a portion of a Ferroportin1 and having at leastone function of a Ferroportin1, as described herein. To producepolyclonal antibodies, the immunogen is introduced into an animal thatis not the original source of the immunogen (e.g., mouse Ferroportin1 ora fragment thereof injected into a non-murine animal).

The term antibody is also intended to encompass single chain antibodies,chimeric, humanized or primatized (CDR-grafted) antibodies and the like,as well as chimeric or CDR-grafted single chain antibodies, comprisingportions from more than one species. For example, the chimericantibodies can comprise portions of proteins derived from two differentspecies, joined together chemically by conventional techniques orprepared as a single contiguous protein using genetic engineeringtechniques (e.g., DNA encoding the protein portions of the chimericantibody can be expressed to produce a contiguous protein chain. See,e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., EuropeanPatent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss etal., European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1;Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400B1; Queen et al., U.S. Pat. No. 5,585,089; and Queen et al., EuropeanPatent No. EP 0 451 216 B1. See also, Newman, R. et al., BioTechnology,10:1455-1460 (1992), regarding primatized antibody, and Ladner et al.,U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science, 242:423-426(1988) regarding single chain antibodies.)

Whole antibodies and biologically functional fragments thereof are alsoencompassed by the term antibody. Biologically functional antibodyfragments which can be used include those fragments sufficient forbinding of the antibody fragment to a Ferroportin1 to occur, such as Fv,Fab, Fab′ and F(ab′)₂ fragments. Such fragments can be produced byenzymatic cleavage or by recombinant techniques. For instance, papain orpepsin cleavage can generate Fab or F(ab′)₂ fragments, respectively.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a chimeric gene encodinga F(ab′)₂ heavy chain portion can be designed to include DNA sequencesencoding the CH₁ domain and hinge region of the heavy chain.

Preparation of Immunizing Antigen (for Instance, Whole Cells Comprisinga Ferroportin1 on the cell surface, or a purified Ferroportin1), andpolyclonal and monoclonal antibody production can be performed using anysuitable technique. A variety of methods have been described for theproduction of antibodies (See e.g., Kohler et al., Nature, 256:495-497(1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature266.550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow,E. and D. Lane, 1988, Antibodies. A Laboratory Manual, (Cold SpringHarbor Laboratory: Cold Spring Harbor, N.Y.); Chapter 11 In CurrentProtocols In Molecular Biology, Vol. 2 (containing supplements upthrough Supplement 49, 2000), Ausubel, F. M. et al., eds., John Wiley &Sons: New York, N.Y.). Generally, a hybridoma can be produced by fusinga suitable immortal cell line (e.g., a myeloma cell line such as SP2/0)with antibody producing cells. The antibody producing cells, preferablythose obtained from the spleen or lymph nodes, can be obtained fromanimals immunized with the antigen of interest. Immunization of animalscan be, for instance, by introduction of whole cells comprisingFerroportin1 protein on the cell surface. The fused cells (hybridomas)can be isolated using selective culture conditions, and cloned bylimiting dilution. Cells which produce antibodies with the desiredspecificity can be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies (includinghuman antibodies) of the requisite specificity can used, including, forexample, methods which select recombinant antibody from a library (e.g.,Hoogenboom et al., WO 93/06213; Hoogenboom et al., U.S. Pat. No.5,565,332; WO 94/13804, published Jun. 23, 1994; and Dower, W. J. etal., U.S. Pat. No. 5,427,908), or which rely upon immunization oftransgenic animals (e.g., mice) capable of producing a full repertoireof human antibodies (see e.g., Jakobovits et al., Proc. Natl. Acad. Sci.USA, 90: 2551-2555 (1993); Jakobovits et al., Nature, 362:255-258(1993); Lonberg et al., U.S. Pat. No. 5,569,825; Lonberg et al., U.S.Pat. No. 5,545,806; Surani et al., U.S. Pat. No. 5,545,807; andKucherlapati, R. et al., European Patent No. EP 0 463 151 B1).

The invention also relates to compositions comprising a modulator ofFerroportin1 function. The term “modulate” as used herein refers to theability of a molecule to alter the function of another molecule. Thus,modulate could mean, for example, inhibit, antagonize, agonize,upregulate, downregulate, induce, or suppress. A modulator has thecapability of altering function of its target. Such alteration can beaccomplished at any stage of the transcription, translation, expressionor function of the protein, so that, for example, modulation of a targetgene can be accomplished by modulation of the DNA or RNA encoding theprotein, and the protein itself.

Antagonists or agonists (inhibitors or enhancers) of the Ferroportin1proteins of the invention, antibodies that bind a Ferroportin1, ormimetics of a Ferroportin1 or of portions of a Ferroportin1 can beemployed in combination with a non-sterile or sterile carrier orcarriers for use with cells, tissues or organisms, such as apharmaceutical carrier suitable for administration to a mammaliansubject. Such compositions comprise, for instance, a media additive or atherapeutically effective amount of an inhibitor or enhancer compound tobe identified by an assay of the invention and a pharmaceuticallyacceptable carrier or excipient. Such carriers may include, but are notlimited to, saline, buffered saline, dextrose, water, ethanol,surfactants, such as glycerol, excipients such as lactose andcombinations thereof. The formulation can be chosen by one of ordinaryskill in the art to suit the mode of administration. The chosen route ofadministration will be influenced by the predominant tissue or organlocation of the Ferroportin1 wherein it is intended that function is tobe inhibited or enhanced. For example, for affecting the function offerroportin1 in the duodenum, a particular administration can be oral orthrough a tube inserted into the stomach (e.g., direct stomach tube ornasopharyngeal tube), or through other means to accomplish delivery tothe small intestine. The invention further relates to diagnostic andpharmaceutical packs and kits comprising one or more containers filledwith one or more of the ingredients of the aforementioned compositionsof the invention.

Compounds of the invention which are Ferroportin1 proteins, Ferroportin1fusion proteins, Ferroportin1 mimetics, ferroportin1 gene-specificantisense poly- or oligonucleotides, inhibitors or enhancers of aFerroportin1 may be employed alone or in conjunction with othercompounds, such as therapeutic compounds. The pharmaceuticalcompositions may be administered in any effective, convenient manner,including administration by topical, oral, anal, vaginal, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal, transdermal orintradermal routes, among others. In therapy or as a prophylactic, theactive agent may be administered to an individual as an injectablecomposition, for example as a sterile aqueous dispersion, preferablyisotonic.

Alternatively, the composition may be formulated for topicalapplication, for example, in the form of ointments, creams, lotions, eyeointments, eye drops, ear drops, mouthwash, impregnated dressings andsutures and aerosols, and may contain appropriate conventionaladditives, including, for example, preservatives, solvents to assistdrug penetration, and emollients in ointments and creams. Such topicalformulations may also contain compatible conventional carriers, forexample cream or ointment bases, and ethanol or oleyl alcohol forlotions.

In addition, the amount of the compound will vary depending on the size,age, body weight, general health, sex, and diet of the recipient of thecompound, and the time of administration, the biological half-life ofthe compound, and the particular characteristics and symptoms of thedisorder to be treated. Adjustment and manipulation of established doseranges are well within the ability of those of skill in the art.

A further aspect of the invention is a method to identify apolymorphism, or the presence of an alternative or variant allele of agene in the genome of an organism (of interest here, genes encodingFerroportin1 proteins). As used herein, polymorphism refers to theoccurrence of two or more genetically determined alternative sequencesor alleles in a population. A polymorphic locus may be as small as abase pair. Polymorphic markers include restriction fragment lengthpolymorphisms, variable number of tandem repeats (VNTR's), hypervariableregions, minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats, simple sequence repeats, and insertion elementssuch as Alu. The first identified alleleic form, or the most frequentlyoccurring form can be arbitrarily designated as the reference (usually,“wildtype”) form, and other allelic forms are designated as alternative(sometimes, “mutant” or “variant”). Diploid organisms may be homozygousor heterozygous for allelic forms.

An “allele” or “allelic sequence” is an alternative form of a gene whichmay result from at least one mutation in the nucleotide sequence.Alleles may result in altered mRNAs or polypeptides whose structure orfunction may or may not be altered. Any given gene may have none, one,or many allelic forms (polymorphism). Common mutational changes whichgive rise to alleles are generally ascribed to natural deletions,additions, or substitutions of nucleotides. Each of these types ofchanges may occur alone, or in combination with the others, one or moretimes in a given sequence.

Several different types of polymorphisms have been reported. Arestriction fragment length polymorphism (RFLP) is a variation in DNAsequence that alters the length of a restriction fragment (Botstein etal., Am. J. Hum. Genet. 32:314-331 (1980)). The restriction fragmentlength polymorphism may create or delete a restriction site, thuschanging the length of the restriction fragment. RFLPs have been widelyused in human and animal genetic analyses (see WO 90/13668; WO 90/11369;Donis-Keller, Cell 51:319-337 (1987); Lander et al., Genetics 121:85-99(1989)). When a heritable trait can be linked to a particular RFLP, thepresence of the RFLP in an individual can be used to predict thelikelihood that the individual will also exhibit the trait.

Other polymorphisms take the form of short tandem repeats (STRs) thatinclude tandem di-, tri- and tetra-nucleotide repeated motifs. Thesetandem repeats are also referred to as variable number tandem repeat(VNTR) polymorphisms. VNTRs have been used in identity and paternityanalysis (U.S. Pat. No. 5,075,217; Armour et al., FEBS Lett. 307:113-115(1992); Horn et al., WO 91/14003; Jeffreys, EP 370,719), and in a largenumber of genetic mapping studies.

Other polymorphisms take the form of single nucleotide variationsbetween individuals of the same species. Such polymorphisms are far morefrequent than RFLPs, STRs (short tandem repeats) and VNTRs (variablenumber tandem repeats). Some single nucleotide polymorphisms occur inprotein-coding sequences, in which case, one of the polymorphic formsmay give rise to the expression of a defective or other variant proteinand, potentially, a genetic disease. Other single nucleotidepolymorphisms occur in noncoding regions. Some of these polymorphismsmay also result in defective protein expression (e.g., as a result ofdefective splicing). Other single nucleotide polymorphisms have nophenotypic effects.

Many of the methods described below require amplification of DNA fromtarget samples and purification of the amplified products. This can beaccomplished by PCR, for instance. See generally, PCR Technology,Principles and Applications for DNA Amplification (ed. H. A. Erlich),Freeman Press, New York, N.Y., 1992; PCR Protocols: A Guide to Methodsand Applications (eds. Innis, et al.), Academic Press, San Diego,Calif., 1990; Mattila et al., Nucleic Acids Res. 19:4967 (1991); Eckertet al., PCR Methods and Applications 1:17 (1991); PCR (eds. McPherson etal., IRS Press, Oxford); and U.S. Pat. No. 4,683,202.

Other suitable amplification methods include the ligase chain reaction(LCR) (see Wu and Wallace, Genomics 4:560 (1989); Landegren et al.,Science 241:1077 (1988)), transcription amplification (Kwoh et al.,Proc. Natl. Acad. Sci. USA 86:1173 (1989), self-sustained sequencereplication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874 (1990),and nucleic acid based sequence amplification (NASBA). The latter twoamplification methods involve isothermal reactions based on isothermaltranscription, which produce both single stranded RNA (ssRNA) and doublestranded DNA (dsDNA) as the amplification products in a ratio of about30 or 100 to 1, respectively.

Another aspect of the invention is a method for detecting a variantallele of a human ferroportin1 gene, comprising preparing amplified,purified ferroportin1 DNA from a reference human and amplified,purified, ferroportin1 DNA from a “test” human to be compared to thereference as having a variant allele, using the same or comparableamplification procedures, and determining whether the reference DNA andtest DNA differ in DNA sequence in the ferroportin1 gene, whether in acoding or a noncoding region, wherein, if the test DNA differs insequence from the reference DNA, the test DNA comprises a variant alleleof a human ferroportin1 gene. The following is a discussion of some ofthe methods by which it can be determined whether the referenceferroportin1 DNA and test ferroportin1 DNA differ in sequence.

Direct Sequencing. The direct analysis of the sequence of variantalleles of the present invention can be accomplished using either thedideoxy chain termination method or the Maxam and Gilbert method (seeSambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed., ColdSpring Harbor Press, New York 1989; Zyskind et al., Recombinant DNALaboratory Manual, Acad. Press, 1988).

Denaturing Gradient Gel Electrophoresis. Amplification productsgenerated using the polymerase chain reaction can be analyzed by the useof denaturing gradient gel eletrophoresis. Different alleles can beidentified based on the different sequence-dependent strand dissociationproperties and electrophoretic migration of DNA in solution (chapter 7in Erlich, ed. PCR Technology, Principles and Applications for DNAAmplification, W.H. Freeman and Co., New York, 1992).

Single-strand Conformation Polymorphism Analysis. Alleles of targetsequences can be differentiated using single-strand conformationpolymorphism analysis, which identifies base differences by alterationin electrophoretic migration of single stranded PCR products, asdescribed in Orita et al., Proc. Natl. Acad. Sci. USA 86:2766-2770(1989). Amplified PCR products can be generated as described above, andheated or otherwise denatured, to form single-stranded amplificationproducts. Single-stranded nucleic acids may refold or form secondarystructures which are partially dependent on the base sequence. Thedifferent electrophoretic mobilities of single-stranded amplificationproducts can be related to base-sequence differences between alleles oftarget sequences.

Detection of Binding by Protein That Binds to Mismatches. Amplified DNAcomprising the ferroportin1 gene or portion of the gene of interest fromgenomic DNA, for example, of a normal individual is prepared, usingprimers designed on the basis of the DNA sequences provided herein.Amplified DNA is also prepared, in a similar manner, from genomic DNA ofan individual to be tested for bearing a distinguishable allele. Theprimers used in PCR carry different labels, for example, primer 1 withbiotin, and primer 2 with ³²P. Unused primers are separated from the PCRproducts, and the products are quantitated. The heteroduplexes are usedin a mismatch detection assay using immobilized mismatch binding protein(MutS) bound to nitrocellulose. The presence of biotin-labeled DNAwherein mismatched regions are bound to the nitrocellulose via MutSprotein, is detected by visualizing the binding of streptavidin tobiotin. See WO 95/12689. MutS protein has also been used in thedetection of point mutations in a gel-mobility-shift assay (Lishanski,A. et al., Proc. Natl. Acad. Sci. USA 91:2674-2678 (1994)).

Other methods, such as those described below, can be used to distinguisha ferroportin1 allele from a reference allele, once a particular allelehas been characterized as to DNA sequence.

Allele-specific probes. The design and use of allele-specific probes foranalyzing polymorphisms is described by e.g., Saiki et al., Nature324:163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548.Allele-specific probes can be designed so that they hybridize to asegment of a target DNA from one individual but do not hybridize to thecorresponding segment from another individual due to the presence ofdifferent polymorphic forms in the respective segments from the twoindividuals. Hybridization conditions should be sufficiently stringentthat there is a significant difference in hybridization intensitybetween alleles, and preferably an essentially binary response, wherebya probe hybridizes to only one of the alleles. Some probes are designedto hybridize to a segment of target DNA such that the polymorphic sitealigns with a central position (e.g., in a 15-mer at the 7 position; ina 16-mer, at either the 8 or 9 position) of the probe. This design ofprobe achieves good discrimination in hybridization between differentallelic forms.

Allele-specific probes are often used in pairs, one member of a pairshowing a perfect match to a reference form of a target sequence and theother member showing a perfect match to a variant form. Several pairs ofprobes can then be immobilized on the same support for simultaneousanalysis of multiple polymorphisms within the same target sequence.

Allele-specific Primers. An allele-specific primer hybridizes to a siteon target DNA overlapping a polymorphism, and only primes amplificationof an allelic form to which the primer exhibits perfect complementarity.See Gibbs, Nucleic Acid Res. 17:2427-2448 (1989). This primer is used inconjunction with a second primer which hybridizes at a distal site.Amplification proceeds from the two primers, resulting in a detectableproduct which indicates the particular allelic form is present. Acontrol is usually performed with a second pair of primers, one of whichshows a single base mismatch at the polymorphic site and the other ofwhich exhibits perfect complementarity to a distal site. The single-basemismatch prevents amplification and no detectable product is formed. Themethod works best when the mismatch is included in the 3′-most positionof the oligonucleotide aligned with the polymorphism because thisposition is most destabilizing to elongation from the primer (see, e.g.,WO 93/22456).

Gene Chips. Allelic variants can also be identified by hybridization tonucleic acids immobilized on solid supports (gene chips), as described,for example, in WO 95/11995 and U.S. Pat. No. 5,143,854, both of whichare incorporated herein by reference. WO 95/11995 describes subarraysthat are optimized for detection of a characterized variant allele. Sucha subarray contains probes designed to be complementary to a secondreference sequence, which is an allelic variant of the first referencesequence.

EXAMPLES

Methods

Zebrafish Strains and Studies

Linkage analysis was performed on haploid or diploid embryos obtainedfrom AB/DAR, AB/SJD or AB/WIK hybrids (Westerfield, M., The ZebrafishBook, Univ. Oregon Press, Eugene, 1993). Wright-Giemsa and o-dianisidinestaining of embryos were performed as described (Ransom, D. G., et al.,Development, 123:311-319, 1996). In situ hybridization analysis wasperformed as described (Thompson, M. A., et al., Dev. Biol.,197:248-269, 1998).

Genetic Mapping and Genotyping and Library Screens

Linkage to centromeric markers (Knapik, E. W., et al., Nature Genet.,18:338-343, 1998) was performed by half-tetrad analysis (Johnson, S. L.,et al., Genetics, 139:1727-1735, 1995). For fine genetic mapping,haploids were genotyped on the proximal side of the locus with one ofthe following RAPD markers (Operon Technologies, Alemeda, Calif.);4W1600, 6Q1300, or 4AC800, and on the distal side with the markers4K1300 or 061020. Diploid mutant embryos were genotyped on the proximalside with the microsatellite markers z8505 or z9479 and on the distalside with z8363 (Shimoda, N., et al., Genomics, 58:219-232, 1999).Library screens were performed as described (Brownlie, A., et al.,Nature Genet., 20:244-250, 1998).

In situ hybridization and rescue experiment embryos (see below) weregenotyped using allele specific oligonucleotide (ASO) hybridizationassays specific to the weh^(tp85c) and weh^(th238) ferroportin1 alleles(Farr, C. J., et al., Proc. Natl. Acad. Sci. USA, 85:1629-1633, 1988;Wood, W. I., et al., Proc. Natl. Acad. Sci. USA, 82:1585-1588, 1985).The weh^(th238) oligonucleotides were developed to distinguish the C toA mutation in the weh^(th238) allele from the wild-type allele.Wild-type is 5′-AAAGAAGTGCGGCCTCATC-3′ (SEQ ID NO:8) and mutantweh^(th238) allele is 5′-AAAGAAGTGAGGCCTCATC-3′ (SEQ ID NO: 9). Theweh^(th85c) oligonucleotides were developed to distinguish the G to Tmutation in the weh^(tp85c) allele from wild-type. Wild-type is5′-GAGCAAATTGGCAGGTAAG-3′ (SEQ ID NO:10) and mutant Weh^(tp85c) alleleis 5′-GAGCAAATTTGCAGGTAAG-3′ (SEQ ID NO:11).

Isolation of the Mouse and Human Ferroportin1 cDNAs

EST clones were identified that contained the 5′ end (GenBank accession# D632209) and 3′ end (GenBank accession # W23461) of human ferroportin1and the 3′ end of mouse ferroportin1 (GenBank accession #AA500296). Thecoding region of human and mouse ferroportin1 cDNAs were cloned byRT-PCR with a forward primer made to the conserved iron response element(IRE) sequence in the 5′ untranslated region(5′-CAACTTCAGCTACAGTGTTAG-3′ (SEQ ID NO:12)) and a reverse primer just3′ of the stop codon of each cDNA (mouse 5′-TTATACAACAGATGTATTCGGT-3′(SEQ ID NO:13) and human 5′-AACTGTCTCAAACAACAGATG-3′ (SEQ ID NO:14)).

Embryo Injection Experiments

A zebrafish Ferroportin1-GFP fusion protein construct was created byPCR. The forward PCR primer contained the start codon,5′-CCGCTCGAGAACGCACAATGGACAGCCCTG-3′ (SEQ ID NO:15). The reverse primercontained the last codon, 5′-CCGCTCGAGTACAGAGTTTGGAAGTGAGGG-3′ (SEQ IDNO:16). The PCR product was subcloned into the GFP expression vectorpEGFP-N1 (Clontech, Palo Alto, Calif.). Embryos from a cross of twoweh^(th238) heterozygotes were injected (Westerfield, M., The ZebrafishBook, Univ. Oregon Press, Eugene, 1993) with the ferroportin1-GFPplasmid (300 ng/ml). For the iron-dextran rescue experiment, 48 hrmutant embryos from a weh^(tp85c) cross were injected intravenously withan iron-dextran solution (Sigma, 100 mg/ml).

Xenopus Oocyte Injections and ⁵⁵Fe Efflux Experiments

cRNA for injection was prepared using the mMessage Machine kit (Ambion,Austin, Tex.), using a construct containing either the rat DMT1 cDNA inpSPORT1 (gift from Hiromi Gunshin (Gunshin, H., et al., Nature,388:482-488, 1997)) or the zebrafish ferroportin1 cDNA in the vectorpXT7 (kindly provided by Sergei Sokol). Defollicularized oocytes wereincubated in ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mMHepes, and 2.5 mM sodium pyruvate, pH 7.4) and injected with either 20ng of DMT1 cRNA alone or 20 ng each of both the DMT1 and ferroportin1cRNAs. ⁵⁵Fe uptake and efflux were performed 48 hrs after injection.⁵⁵Fe uptake was performed in 500 μl of a solution containing 60 μM⁵⁵FeCl₂, 100 mM NaCl, 10 mM Hepes and 1 mM ascorbic acid at pH 5.5 for30 minutes. ⁵⁵Fe uptake was stopped by incubation of the oocytes in 1 mMcold FeCl₂, 100 mM NaCl, 10 mM Hepes, and 1 mM ascorbic acid at pH 6.0for 30 minutes. Individual oocytes were placed in 500 μl of eitherefflux buffer (100 mM NaCl, 10 mM Hepes at pH 7.4) alone or effluxbuffer containing a final concentration of 20 mg/ml apo-transferrin for60 minutes. After incubation, the ⁵⁵Fe levels of both the effluxsolution and the individual oocytes lysed in 10% SDS were measured byscintillation counting.

Mouse In Situ Hybridization and Northern Blot Analysis

In situ hybridization of murine embryos was performed as described(Palis, J., et al., Mol. Reprod. Dev., 42:19-27, 1995). An adultmulti-tissue Human Northern blot (Clontech, Palo Alto Calif.) containing2 μg of poly A⁺ mRNA per lane was probed with a human ferroportin1 EST(GenBank accession #WO5488). The mouse intestinal Northern blotcontaining 20 μg of total RNA per lane was probed with a mouseferroportin1 EST (GenBank accession #AA500296).

Antibody Generation and Immunohistochemistry

A rabbit polyclonal antibody was generated to a peptide consisting ofthe C-terminal 19 amino acids of the human Ferroportin1 protein (GenemedSynthesis, San Francisco Calif.). Antiserum was affinity-purifiedagainst the peptide. Formalin or Bouin's fixed paraffin-embeddedspecimens were deparaffinized and heat treated for 30 min in 1.0 mMEDTA, pH 8.0, in a Black and Decker steamer (Model HS80). Endogenousperoxidase activity was quenched in methanol and 3% hydrogen peroxide(5:1, vol/vol). The slides were then incubated in 3% normal swine serumin 0.05 M Tris, pH 7.6, followed by rabbit anti-Ferroportin1 antibody (7μg/ml), and sequentially in HRP-conjugated goat anti-rabbitimmunoglobulins (1:40 dilution, Dako), HRP-conjugated rabbit anti-goatimmunoglobulins (1:40 dilution, Dako), followed by HRP-conjugated swineanti-rabbit immunoglobulins (1:52 dilution, Dako), each prepared in 0.10M Tris, pH 7.6, containing (4%) human AB serum. Antibody localizationwas determined using DAB in 0.5 M Tris (pH 7.6) containing (0.035%)hydrogen peroxide. Slides were counterstained with methyl green. Controlsamples were incubated with normal rabbit serum or purified rabbitimmunoglobulins at a protein concentration equal to the antibodypreparation. In addition, preincubation of the antibody with specificpeptide at a 550-fold molar excess neutralized the reactivity.

Red Cell Iron Measurement

Iron levels in red blood cells were measured by Atomic AbsorptionSpectrometer Model 3030 equipped with Zeeman Graphite Furnace andAutosampler, (Perkin Elmer Corp.). A cell sample (at least 30,000 cellsin 20 □l PBS) or blank (PBS) was diluted with 40 μl of 480 mg/dl ofmagnesium nitrate (matrix modifier); 20 μl of the mixture was injectedinto the instrument and analyzed in duplicate. The instrument wascalibrated with iron standards of 10, 25, 100 and 250 ng/ml preparedfrom Atomic Spectroscopy Standard (Perkin Elmer Corp).

Structure Prediction

Hydropathy plots (Kyte-Doolittle) were obtained using the GeneticsComputer Group (GCG) programs PEPTIDESTRUCTURE and PEPPLOT with anhydropathy window of 14. Transmembrane amino acid segments wereidentified and their topography predicted using the programs PHDhtm(www.embl-heidelberg.de/predictprotein/predictprotein.html), HMMTOP(www.enzim.hu/hmmtopi), TMHMM (www.cbs.dtu.dk/services/TMHMM-1.0/),TMpred (www.ch.embnet.org/software/TMPRED_form.html), TopPred2(www.biokemi.su.se/˜server/toppred2/), and SOSUI(www.tuat.ac.jp/˜mitaku/). Most analyses predict 10 TM segments. Somepredict that the first and last TM segments are split, and thatFerroportin contains 12 segments. Others predict that TM segments 4 andare not split and only 9 segments are present.

Example 1 Observations on Weissherbst Zebrafish Mutants

Two independent autosomal recessive mutations of the zebrafishhypochromic blood mutant weissherbst (weh), weh^(th238) and weh^(tp85c),were isolated as part of a large-scale screen for ethyl nitroso urea(ENU) induced mutations that disrupt embryonic development in zebrafish(Haffter, P., et al., Danio rerio. Development, 123:1-36, 1996). Theseweh mutants were identified in a morphologic screen for defects incirculating erythroid cells (Ransom, D. G., et al., Development,123:311-319, 1996). While the number of circulating erythroid cells ofboth mutant alleles are normal at 33 and 48 hours post fertilization(hpf), the weh mutant cells are hypochromic (lacking red color). Mutantembryos show little, if any, hemoglobin compared to wild-types at 33 hpfand 48 hpf (by o-dianisidine staining), but some hemoglobin isdetectable at 72 hpf. The weh^(th238) allele has less o-dianisidinestaining compared to the weh^(tp85c) allele at 72 hpf, suggesting thatit is a more severely defective allele. In addition to the hypochromia,a progressive decrease in red cell number occurs after 48 hpf. By 96hpf, the number of circulating erythroid cells in mutants decreases toapproximately 20% of the number in wild-type. The weh mutant cells atdays 2 (56 hpf) and 3 (80 hpf) have a large nucleus and basophiliccytoplasm characteristic of more immature erythroid cells, and on day 5(125 hpf) the remaining mutant cells are misshapen. Further studies oferythroid differentiation reveal that embryonic globin mRNA levels areabnormally maintained during maturation. Although weh mutants have nogross organ defects in addition to their anemia, all mutant embryos diebetween day 7 and day 14 of development.

Given the possibility that hypochromia could result from irondeficiency, we measured iron levels in weh erythroid cells. The level ofiron measured in 10⁴ wild-type cells ranged from 0.093 ng to 0.208 ng(n=3), whereas the levels of iron in the same number of weh mutant cellsranged from 0.014 ng to 0.033 ng (n=3). The 4-9 fold decrease inerythrocyte iron levels could be due to low levels of iron incirculation. As confirmation of this hypothesis, iron-dextran injectedintravenously into weh^(tp85c) mutant embryos was shown to completelyrescue hemoglobin production as seen by o-diansidine staining ofhemoglobin. This rescue demonstrated that weh mutant erythroid cells arefully capable of hemoglobinization, and that the hypochromia is causedby inadequate circulatory iron levels.

Example 2 Isolation of weh Mutant Gene

To gain further insight into this phenotype, we isolated the weh mutantgene by positional cloning methods. Study of the segregation ofcentromeric microsatellite markers in half-tetrad gynogenetic diploidembryos localized weh to linkage group 9. Genetic mapping placed the wehlocus in an approximately 6 cM interval between the random amplifiedpolymorphic DNA (RAPD) markers 4K1300 and 6Q1300 (FIG. 1). We isolatedmore closely linked markers using amplified fragment length polymorphism(AFLP) analysis (Ransom, D. G., et al., pp. 195-210 in The Zebrafish:Genetics and Genomics, eds. Detrich, H. W. L., Westerfield, M. & Zon, L.I., Academic, San Diego, 1999; Vos. P., et al., Nucleic Acids Res.,23:4407-4414, 1995), scanning approximately 10,000 polymorphic loci.Single strand conformational polymorphism (SSCP) analysis showed thatthe AFLP marker 136 was 0.13 cM proximal to the weh locus (FIG. 1).

A chromosomal walk towards the gene was initiated from the I36 marker(FIG. 1), resulting in the identification of a critical interval thatcontained the weh gene (FIG. 1). In an attempt to identify potentialcandidates for the weh gene, we used a hybridization strategy to screencDNA libraries for genes located on the PAC clones identified in theregion of the weh locus. We hybridized a radiolabelled insert of PACclone 170G3 to zebrafish gridded cDNA libraries. Screening of 100,000gridded cDNA clones identified 5 clones of a novel cDNA, designated WC1for weh cDNA #1. Analysis of WC1 mRNA expression in embryos andsequencing WC1 from weh^(th238) mutants suggested that WC1 was not acandidate for weh. Two genes, STAT1 and glutaminase, were isolated byhybridization of the zebrafish PAC clone 8714 (FIG. 1) to gridded cDNAlibraries. The human orthologs of WC1, STAT1 and glutaminase arelocalized to human chromosome 2 in a 2.4 cM interval, demonstratingconserved chromosomal synteny among vertebrates (Postlethwait, J. H., etal., Nature Genet., 18:345-349, 1998). Homology searches identified apufferfish (Fugu rubripes) cosmid clone (121D21) that contained both WC1and STAT1. This cosmid also contained Fugu homologs of other geneslocated on human chromosome 2. Using primers designed to the pufferfishsequence of one of these genes (121D21 aB3), a 200 bp fragment of thezebrafish ortholog was amplified from PAC 211013. A full length cDNA(3.7 kb) of this gene, hereafter referred to as ferroportin1, wasisolated from a zebrafish kidney cDNA library.

This gene, ferroportin1, has a predicted open reading frame of 562 aminoacids (FIG. 2). Sequence analysis of the weh^(th238) allele identified aC to A nucleotide transversion that causes premature termination oftranslation at codon 361 (FIG. 2). Similar analysis of the weh^(tp85c)allele identified a single amino acid change, Leu168Phe, resulting froma G to T nucleotide difference (FIG. 2). The finding of a premature stopmutation in weh^(th238) strongly suggests that the weh mutant phenotypeis caused by a defect in ferroportin1. Mouse and human ferroportin1 cDNAclones were obtained by RT-PCR of RNA isolated from liver and placenta,respectively. A conserved sequence, predicted to form a hairpin loopstructure typical of iron response elements (IREs) (Eisenstein, R. S.,et al., J. Nutr., 128:2295-2298, 1998), was identified in the 5′untranslated region (UTR) of the cDNAs from all three species. Based onprotein structure prediction analysis, Ferroportin1 contains at least 10transmembrane segments (FIG. 2).

Example 3 Expression of Ferroportin1

In situ hybridization analysis of zebrafish embryos shows thatferroportin1 mRNA is not expressed in erythroid cells. Ferroportin1 mRNAis detected at 18 hpf through 48 hpf in the yolk syncytial layer (YSL).The YSL is the peripheral layer of the yolk cell that lies just belowthe membrane (Kimmel, C. B., et al., Dev. Dyn., 203:253-310, 1995). Thislayer surrounds the entire yolk of the embryo and consists of yolk-freecytoplasm and nuclei. Yolk has been shown to contain nutrients neededduring development (Richards, M. P., Poult. Sci., 76:152-164, 1997),including iron (Richards, M. P., Poult. Sci., 76:152-164, 1997; Dumont,J. N., J. Exp. Zool., 204:193-217, 1978; Craik, J. C., Comp. Biochem.Physiol. A., 83:515-517, 1986). Embryos express ferroportin1 in theregion of the YSL at 18 hpf that lies just below the developinghematopoietic cells in the intermediate cell mass (Al-Adhami, M. A., etal., Develop. Growth Differ., 19:171-179, 1977). At 48 hpf, ferroportin1is expressed in the brain and in a localized region of the YSL. Notethat expression is in the same region of the YSL over which the bloodflows (Reib, J. P., Annales D'Embryologie et de Morphogenese, 6:43-54,1973). At both time-points, ferroportin1 is expressed in the region ofthe YSL adjacent to the blood, but not by the entire YSL. This patternof expression suggests that ferroportin1 expression and function in theYSL is linked to red blood cell development. Considering theiron-dextran rescue of hemoglobin production in weh mutants, the YSLexpression of ferroportin1 suggested that the gene might function in thetransport of iron from the yolk to the embryonic circulation.

To provide evidence that defects in the ferroportin1 gene cause the wehmutant phenotype, we injected a plasmid that expresses Ferroportin1fused to green fluorescent protein (GFP) into the yolk cell between the256 and 1000 cell stages (Kimmel, C. B., et al., Dev. Dyn., 203:253-310,1995). At 48 hours of development, 33% of injected embryos expressed GFPstrictly in the YSL. At 80 hpf, the phenotype of mutant embryosexpressing GFP was compared to the uninjected mutants. TheFerroportin1-GFP-expressing mutant embryos (n=9) had considerably morehemoglobin expression than uninjected mutants as observed byo-dianisidine staining. This partial rescue of the hypochromia providesfurther evidence that ferroportin1 is the weh gene, and demonstratesthat Ferroportin1 acts in the YSL. The rescue of the weh mutantphenotype by intravenous iron-dextran injection and by Ferroportin1expressed in the YSL indicates that Ferroportin1 functions to deliveryolk iron into the embryonic circulation.

The function of Ferroportin1 was tested using a Xenopus oocyteexpression system. Since the proposed function of Ferroportin1 is toexport iron, it was necessary to first load the oocytes with ⁵⁵Fe.Radioactive iron loading was accomplished through the expression of theiron transporter DMT1 in oocytes and then loading of ⁵⁵Fe at pH 5.5.⁵⁵Fe loaded oocytes that expressed either DMT1 alone, or DMT1 andFerroportin1, were tested for iron export activity either in thepresence or absence of apo-transferrin, an iron chelator. To normalizefor the iron content in each individual oocyte, the ratio of efflux touptake was calculated. Our results showed that, in the presence ofapo-transferrin, the efflux to uptake ratio in oocytes expressingFerroportin1 was five fold greater than control oocytes not expressingFerroportin1 (FIG. 3, P<<0.001).

Example 4 Expression of Ferroportin1 in Embryonic and Adult Tissues ofMammals

To evaluate a potential role for ferroportin1 in iron transport inmammals, we examined tissue expression. Northern blot analysis showedhighest levels of expression in human placenta, liver, spleen, andkidney. In mice, ferroportin1 mRNA is expressed specifically in theduodenum but not in the jejunum or ileum. Additionally, ferroportin1 isexpressed in the large intestine. Most intestinal iron absorption occursin the proximal duodenum, placing ferroportin1 in a physiologicallyappropriate location to play a role in intestinal iron absorption. mRNAin situ hybridization analysis was performed on sections of mouseembryos. The primitive erythroblasts derived from the blood islands donot express Ferroportin1, whereas the trophoblast cells of the innerplacenta express high levels of ferroportin1 RNA. Ferroportin1transcripts were found in the inner placenta (labyrinth zone) and thetrophoblast giant cells at the border between the outer placenta(spongiotrophoblast), but not in the maternal deciduum. Ferroportin1expression is also present in the visceral endoderm of the yolk sacsurrounding the embryo proper. Within the embryo proper, ferroportin1transcripts were detected in several tissues, including the vascularplexus surrounding the central nervous systems, but particularly the gutand liver. The expression of ferroportin1 in placenta, duodenum, andliver, all prominent sites of iron transport, is consistent with theproposed role of the gene in iron transport.

In order to characterize the expression of Ferroportin1 protein, wegenerated a specific rabbit polyclonal antibody against a Ferroportin1peptide. In the human placenta, Ferroportin1 protein was primarilyexpressed in a basal location within the syncytiotrophoblasts. The basalsurface of the syncytiotrophoblast interfaces with the fetalcirculation, whereas the apical surface contacts the maternalcirculation. The mammalian placenta and the zebrafish YSL provide ahomologous function, serving as the site of iron transfer between motherand embryo. Taken together with the functional data in zebrafish andXenopus, Ferroportin1 is likely to export iron from thesyncytiotrophoblast into the embryonic circulation.

A similar analysis of mouse duodenum showed Ferroportin1 staining inenterocytes in the villus. The intensity of staining was stronger at thetip of the villus compared to the crypt. Staining was particularlystrong at the basolateral surface of the enterocyte. The duodenalenterocytes of the small intestine are polarized epithelial cells thattransport iron into the intestinal capillaries through the basolateralmembrane. The mechanism of intestinal basolateral iron transport has notbeen established. Sex-linked anemia (sla) mice have a defect inbasolateral iron transport in the duodenum, based on ferrokineticstudies and the presence of abnormal iron deposits in duodenalenterocytes (Bannerman, R. M., Fed. Proc., 35:2281-2285, 1976).Analogous to weh mutants, the sla mouse has a defect in transport ofmaternal iron to the embryonic circulation (Kingston, P. J., et al., Br.J. Haematol., 40:265-276, 1978). The sla phenotype is due to a mutationin the membrane-bound multi-copper ferroxidase gene, hephaestin (Vulpe,C. D., et al., Nature Genet., 21:195-199, 1999). In Saccharomycescerevisiae, the hephaestin-like ferroxidase FET3 is required for highaffinity iron uptake by the iron transporter FTR1 (Askwith, C., et al.,Cell, 76:403-410, 1994; Stearman, R., et al., Science, 271:1552-1557,1996). Expression of Ferroportin1 at the basolateral surface of duodenalenterocytes in mouse and the multiple-transmembrane structure of theprotein make it an excellent candidate to function as a basolateral irontransporter.

Additional data from the weh mutant suggests that Ferroportin1 functionsin the intestine of the adult zebrafish. Both in situ hybridizationstudies and immunohistochemistry showed expression of zebrafishferroportin1 in the intestine. In addition, iron-dextran rescued mutantembryos live past the normal time of lethality (day 7-14). We havesuccessfully raised these rescued embryos to adulthood. These fish aresmaller than their wildtype siblings and have a profound hypochromicanemia. Since these fish eat a normal diet replete in iron, butnonetheless are severely anemic, these data suggest that the gene isrequired for intestinal iron absorption in addition to yolk sactransport. Based on the basolateral expression pattern of Ferroportin1in mammalian enterocytes and the implication that ferroportin1 isrequired for intestinal iron transport in zebrafish, it is likely thatthe protein is involved in iron export from enterocytes in mammals.Further experiments are required to determine whether Ferroportin1cooperates with hephaestin in these cells.

Other tissues may utilize ferroportin1 as an iron exporter. Very highlevels of expression are evident in Kupffer cells of human liver, theresident macrophages of the liver, and macrophages located within thesplenic red pulp. Hepatocytes were also positive by immunohistochemicalstaining. Ferroportin1 could play a role in iron export frommacrophages, a critical function in recycling of iron from senescenterythrocytes.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for eliciting an immune response in an animal, said methodcomprising introducing into the animal a composition comprising apolypeptide comprising at least 19 consecutive amino acid residues ofSEQ ID NO:2.
 2. A method for eliciting an immune response in an animal,said method comprising introducing into the animal a compositioncomprising a polypeptide comprising at least 19 consecutive amino acidresidues of SEQ ID NO:4.
 3. A method for eliciting an immune response inan animal, said method comprising introducing into the animal acomposition comprising a polypeptide comprising at least 19 consecutiveamino acid residues of SEQ ID NO:6.
 4. Antibodies that bind specificallyto a Ferroportin1 protein, where antibodies include single-chainantibodies, chimeric antibodies and immunologically active fragments ofantibodies.
 5. A method for producing antibodies, said method comprisingintroducing into an animal isolated zebrafish Ferroportin1 or animmunogenic fragment of zebrafish Ferroportin1.
 6. A method forproducing antibodies, said method comprising introducing into anon-murine animal isolated mouse Ferroportin1 or an immunogenic fragmentof mouse Ferroportin1.
 7. A method for producing antibodies, said methodcomprising introducing into a non-human animal isolated humanFerroportin1 or an immunogenic fragment of human Ferroportin1.